Topical antiviral compositions, delivery systems, and methods of using the same

ABSTRACT

The present invention relates generally to topical antiviral compositions, delivery systems, and methods of using the same.

RELATED APPLICATION INFORMATION

This application is a continuation of U.S. application Ser. No.16/431,214, filed Jun. 4, 2019, which is a continuation of U.S.application Ser. No. 15/713,185, filed Sep. 22, 2017, now U.S. Pat. No.10,322,082, issued Jun. 18, 2019, which is a continuation-in-part ofU.S. application Ser. No. 15/324,332, filed Jan. 6, 2017, now U.S. Pat.No. 10,322,081, issued Jun. 18, 2019, which is a 35 U.S.C. § 371national phase application of International Application Serial No.PCT/US2015/039908, filed Jul. 10, 2015, which claims the benefit of andpriority to U.S. Application Ser. No. 62/023,587, filed Jul. 11, 2014,and U.S. Application Ser. No. 62/139,176, filed Mar. 27, 2015, and U.S.Application Serial No. 15/713,185 is a continuation of InternationalApplication Serial No. PCT/US2016/012668, filed Jan. 8, 2016, whichclaims the benefit of and priority to International Application SerialNo. PCT/US2015/039908, filed Jul. 10, 2015, which claims the benefit ofand priority to U.S. Application Ser. No. 62/023,587, filed Jul. 11,2014, and U.S. Application Serial No. 62/139,176, filed Mar. 27, 2015;and this application is a continuation of U.S. application Ser. No.15/324,332, filed Jan. 6, 2017, which is a 35 U.S.C. § 371 nationalphase application of International Application Serial No.PCT/US2015/039908, filed Jul. 10, 2015, which claims the benefit of andpriority to U.S. Application Ser. No. 62/023,587, filed Jul. 11, 2014,and U.S. Application Ser. No. 62/139,176, filed Mar. 27, 2015; thedisclosure of each of which are incorporated herein by reference intheir entirety.

FIELD

The present invention relates generally to topical antiviralcompositions, delivery systems, and methods of using the same. Deliverysystems may include a topical antiviral composition. Methods of usingthe topical antiviral compositions and/or delivery systems includemethods of treating and/or preventing a viral infection.

BACKGROUND

Viruses cause a number of diseases that can be treated topically. Forexample, warts can be caused by human papillomavirus and can be treatedtopically. However, viruses can be difficult to treat since they invadehost cells and replicate. In addition, new viral strains have emerged,including antiviral resistant strains.

SUMMARY

It is noted that aspects described with respect to one embodiment may beincorporated in different embodiments although not specificallydescribed relative thereto. Some embodiments are directed tocompositions, kits, and/or methods for treating and/or preventing aviral infection. In some embodiments, a method of treating and/orpreventing a viral infection in a subject in need thereof is provided.

In some embodiments, the method includes administering a topicalcomposition to the skin of a subject, wherein the topical compositioncomprises a nitric oxide-releasing active pharmaceutical ingredient inan amount of about 0.5% to about 20% by weight of the composition,thereby treating and/or preventing the viral infection in the subject.

In some embodiments, the method includes administering a topicalcomposition to the skin of a subject, wherein the topical compositioncomprises a nitric oxide-releasing active pharmaceutical ingredient thatreleases nitric oxide to the skin of the subject, and wherein thetopical composition maintains a real time concentration of nitric oxideof at least about 7 pmol of NO/mg of the composition for at least 1 hourafter administration, as measured by real time in vitro release testing,thereby treating and/or preventing the viral infection in the subject.

In some embodiments, the method includes administering a topicalcomposition to the skin of a subject, wherein the topical compositioncomprises a nitric oxide-releasing active pharmaceutical ingredient thatreleases nitric oxide to the skin of the subject, and wherein thetopical composition maintains a real time concentration of nitric oxideof at least about 104 pmol of NO/cm² over a time period of at least 1hour after administration of the composition to the skin of the subject,as measured by real time in vitro release testing, thereby treatingand/or preventing a viral infection in the subject.

The foregoing and other aspects of the present invention will now bedescribed in more detail with respect to other embodiments describedherein. It should be appreciated that the invention can be embodied indifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the outline of experimental infections on a rabbit.

FIG. 2 shows a graph of the mean±SEM of geometric mean diameter (GMD)measurements of CRPV-induced rabbit papillomas from rabbits in Group A.Papillomas were induced at 2 sites with 5 μg of wt-CRPV plasmid stock(●, ▾), and at 2 sites with 5μg mE8-CRPV plasmid stock (◯, Δ). Leftsites (L1 and L2) were treated topically with placebo gel (●, ◯) andright sites (R1 and R2) were untreated (▾, Δ). Each symbol representsthe mean (±SEM) of GMDs of the weekly measurements.

FIG. 3 shows a graph of mean±SEM of GMD measurements of CRPV-inducedrabbit papillomas from rabbits in Group B. Papillomas were induced at 2sites with 5 μg of wt-CRPV plasmid stock (●, ▾), and at 2 sites with 5μg mE8-CRPV plasmid stock (◯, Δ). Left sites (L1 and L2) were treatedtopically with 1% Nitricil™ NVN1 (●, ◯) and right sites (R1 and R2) wereuntreated (▾, Δ). Each symbol represents the mean (±SEM) of GMDs of theweekly measurements.

FIG. 4 shows a graph of mean±SEM of GMD measurements of CRPV-inducedrabbit papillomas from rabbits in Group C. Papillomas were induced at 2sites with 5 μg of wt-CRPV plasmid stock (●, ▾), and at 2 sites with 5μg mE8-CRPV plasmid stock (◯, Δ). Left sites (L1 and L2) were treatedtopically with 1.6% Nitricil™ NVN4 (●, ◯) and right sites (R1 and R2)were untreated (▾, Δ). Each symbol represents the mean (±SEM) of GMDs ofthe weekly measurements.

FIG. 5 shows a graph of mean±SEM of GMD measurements of CRPV-inducedrabbit papillomas from rabbits in Group D. Papillomas were induced at 2sites with 5 μg of wt-CRPV plasmid stock (●, ▾), and at 2 sites with 5μg mE8-CRPV plasmid stock (◯, Δ). Left sites (L1 and L2) were treatedtopically with 10% Nitricil™ NVN1 (●, ◯) and right sites (R1 and R2)were untreated (▾, Δ). Each symbol represents the mean (±SEM) of GMDs ofthe weekly measurements.

FIG. 6 shows a graph of mean±SEM of GMD measurements of CRPV-inducedrabbit papillomas from rabbits in Group E. Papillomas were induced at 2sites with 5 μg of wt-CRPV plasmid stock (●, ▾), and at 2 sites with 5μg mE8-CRPV plasmid stock (◯, Δ). Left sites (L1 and L2) were treatedtopically with 16.3% Nitricil™ NVN4 (●, ◯) and right sites (R1 and R2)were untreated (▾, Δ). Each symbol represents the mean (±SEM) of GMDs ofthe weekly measurements.

FIG. 7 shows a graph of mean±SEM of GMD measurements of CRPV-inducedrabbit papillomas from rabbits in Group F. Papillomas were induced at 2sites with 5 μg of wt-CRPV plasmid stock (●, ▾), and at 2 sites with 5μg mE8-CRPV plasmid stock (◯, Δ). Left sites (L1 and L2) were treatedtopically with placebo ointment (●, ◯) and right sites (R1 and R2) wereuntreated (▾, Δ). Each symbol represents the mean (±SEM) of GMDs of theweekly measurements.

FIG. 8 shows a graph of mean±SEM of GMD measurements of CRPV-inducedrabbit papillomas from rabbits in Group G. Papillomas were induced at 2sites with 5 μg of wt-CRPV plasmid stock (●, ▾), and at 2 sites with 5μg mE8-CRPV plasmid stock (◯, Δ). Left sites (L1 and L2) were treatedtopically with a single phase, 10% Nitricil™ NVN1 ointment (●, ◯) andright sites (R1 and R2) were untreated (▾, Δ). Each symbol representsthe mean (±SEM) of GMDs of the weekly measurements.

FIG. 9 shows a graph of mean±SEM of GMD measurements of CRPV-inducedrabbit papillomas from rabbits in Group H. Papillomas were induced at 2sites with 5 μg of wt-CRPV plasmid stock (●, ▾), and at 2 sites with 5μg mE8-CRPV plasmid stock (◯, Δ). Left sites (L1 and L2) were treatedtopically with 0.3% cidofovir in cremophor (50%) (●, ◯) and right sites(R1 and R2) were untreated (▾, Δ). Each symbol represents the mean(±SEM) of GMDs of the weekly measurements.

FIG. 10 shows a graph of mean±SEM rabbit weight (kg) for rabbits inGroups A-H. Weights are plotted together with SEM error bars againsttime after infection with CRPV.

FIG. 11 shows a graph of the cumulative nitric oxide (NO) release overtime for the formulations used in Groups B, D, and G.

FIG. 12 shows a graph of the real time NO release over time for theformulations used in Groups B and D.

FIG. 13 shows a graph of the cumulative NO release over time for theformulations used in Groups C, E, and G.

FIG. 14 shows a graph of the real time NO release over time for theformulations used in Groups C and E.

FIG. 15 shows a graph of the cumulative NO release over time for theformulations used in Groups B, C, D, E, and G.

FIG. 16A shows a graph of the real time NO release in pmol/mg for theformulations used in Groups B, D, E and G with rectangles representing 1hour, 2 hour, and 4 hour time periods with ranges of NO releaseaccording to some embodiments of the present invention.

FIG. 16B is an enlarged version of the first 1.5 hours of FIG. 16A witha rectangle representing the 1 hour time period with ranges of NOrelease according to some embodiments of the present invention.

FIG. 17 shows a graph of the real time NO release in pmol/cm² for theformulations used in Groups D and E with rectangles representing 1 hour,2 hour, and 4 hour time periods with ranges of NO release per cm²according to some embodiments of the present invention.

FIG. 18 shows a graph of papilloma size (GMD) in mm over time for theformulation used in Group A. Papillomas were induced at 2 sites with 5μg of wt-CRPV plasmid stock (●, ▾), and at 2 sites with 5 μg mE8-CRPVplasmid stock (◯, Δ). Left sites (L1 and L2) were treated topically withthe placebo (●, ◯) and right sites (R1 and R2) were untreated (▾, Δ).Each symbol represents the mean (±SEM) of GMDs of the weeklymeasurements.

FIG. 19 shows a graph of papilloma size (GMD) in mm over time for theformulation used in Group B. Papillomas were induced at 2 sites with 5μg of wt-CRPV plasmid stock (●, ▾), and at 2 sites with 5 μg mE8-CRPVplasmid stock (◯, Δ). Left sites (L1 and L2) were treated topically withthe 2% Nitricil™ NVN1 formulation (●, ◯) and right sites (R1 and R2)were untreated (▾, Δ). Each symbol represents the mean (±SEM) of GMDs ofthe weekly measurements.

FIG. 20 shows a graph of papilloma size (GMD) in mm over time for theformulation used in Group C. Papillomas were induced at 2 sites with 5μg of wt-CRPV plasmid stock (●, ▾), and at 2 sites with 5 μg mE8-CRPVplasmid stock (◯, Δ). Left sites (L1 and L2) were treated topically withthe 4% Nitricil™ NVN1 formulation (●, ◯) and right sites (R1 and R2)were untreated (▾, Δ). Each symbol represents the mean (±SEM) of GMDs ofthe weekly measurements.

FIG. 21 shows a graph of papilloma size (GMD) in mm over time for theformulation used in Group D. Papillomas were induced at 2 sites with 5μg of wt-CRPV plasmid stock (●, ▾), and at 2 sites with 5 μg mE8-CRPVplasmid stock (◯, Δ). Left sites (L1 and L2) were treated topically withthe 8% Nitricil™ NVN1 formulation (●, ◯) and right sites (R1 and R2)were untreated (▾, Δ). Each symbol represents the mean (±SEM) of GMDs ofthe weekly measurements.

FIG. 22 shows a graph of papilloma size (GMD) in mm over time for theformulation used in Group E. Papillomas were induced at 2 sites with 5μg of wt-CRPV plasmid stock (●, ▾), and at 2 sites with 5 μg mE8-CRPVplasmid stock (◯, Δ). Left sites (L1 and L2) were treated topically withthe 10% Nitricil™ NVN1 formulation (●, ◯) and right sites (R1 and R2)were untreated (▾, Δ). Each symbol represents the mean (±SEM) of GMDs ofthe weekly measurements.

FIG. 23 shows a graph of papilloma size (GMD) in mm over time for theformulation used in Group F. Papillomas were induced at 2 sites with 5μg of wt-CRPV plasmid stock (●, ▾), and at 2 sites with 5 μg mE8-CRPVplasmid stock (◯, Δ). Left sites (L1 and L2) were treated topically withthe imiquimod control (●, ◯) and right sites (R1 and R2) were untreated(▾, Δ). Each symbol represents the mean (±SEM) of GMDs of the weeklymeasurements.

FIGS. 24A-24C illustrate representative images of uninfected raftcultures that were treated with 0.5, 0.75, and 1.0 mg/mL of Nitricil™NVN1, respectively, and are stained with H&E or labeled with BrdUantibodies.

FIGS. 25A-25C illustrate representative images of HPV-18 infectedcultures that were treated with 0.75, 1.0, and 1.5 mg/mL of Nitricil™NVN1, respectively, and are stained with H&E or labeled with BrdUantibodies.

FIGS. 26A-26D illustrate representative images of uninfected raftcultures that were treated with 0.75, 1.0, 1.5, and 2.0 mg/mL ofNitricil™ NVN4, respectively, and are stained with H&E or labeled withBrdU antibodies.

FIGS. 27A-27D illustrate representative images of HPV-18 infectedcultures that were treated with 0.75, 1.0, 1.5, and 2.0 mg/mL ofNitricil™ NVN4, respectively, and are stained with H&E or labeled withBrdU antibodies.

DETAILED DESCRIPTION

The present invention will now be described more fully hereinafter. Thisinvention may, however, be embodied in different forms and should not beconstrued as limited to the embodiments set forth herein. Rather, theseembodiments are provided so that this disclosure will be thorough andcomplete, and will fully convey the scope of the invention to thoseskilled in the art.

The terminology used in the description of the invention herein is forthe purpose of describing particular embodiments only and is notintended to be limiting of the invention. As used in the description ofthe invention and the appended claims, the singular forms “a”, “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the present applicationand relevant art and should not be interpreted in an idealized or overlyformal sense unless expressly so defined herein. The terminology used inthe description of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. In case of a conflict in terminology, the presentspecification is controlling.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination. Moreover, the present invention also contemplates thatin some embodiments of the invention, any feature or combination offeatures set forth herein can be excluded or omitted. To illustrate, ifthe specification states that a complex comprises components A, B and C,it is specifically intended that any of A, B or C, or a combinationthereof, can be omitted and disclaimed.

As used herein, the transitional phrase “consisting essentially of” (andgrammatical variants) is to be interpreted as encompassing the recitedmaterials or steps “and those that do not materially affect the basicand novel characteristic(s)” of the claimed invention. See, In re Herz,537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in theoriginal); see also MPEP § 2111.03. Thus, the term “consistingessentially of” as used herein should not be interpreted as equivalentto “comprising.”

The term “about,” as used herein when referring to a measurable value,such as an amount or concentration and the like, is meant to refer tovariations of up to ±20% of the specified value, such as, but notlimited to, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value,as well as the specified value. For example, “about X” where X is themeasurable value, is meant to include X as well as variations of ±20%,±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of X. A range provided herein for ameasureable value may include any other range and/or individual valuetherein.

According to some embodiments of the present invention, provided hereinare methods of treating and/or preventing a viral infection. A method oftreating and/or preventing a viral infection may comprise administeringa topical antiviral composition (i.e., a composition of the presentinvention) to the skin of a subject, thereby treating and/or preventingthe viral infection in the subject. In some embodiments, the topicalantiviral composition may be administered and/or applied to virallyinfected skin of the subject. In some embodiments, a method of thepresent invention may suppress and/or inhibit viral replication of avirus and/or enhance the local immune response of a subject. In someembodiments, administration of the topical antiviral composition to theskin of a subject may provide for topical and/or transdermal delivery ofnitric oxide (NO) to the subject. In some embodiments, a method of thepresent invention may provide for targeted delivery of NO to an area ofskin of a subject and/or may provide for local, systemic delivery of NOto the skin and/or surrounding tissues and/or organs of the subject. Insome embodiments, a method of administering a composition of the presentinvention may administer NO to the skin of a subject and/or through theskin to a localized area.

In some embodiments, a drug delivery system may be used to administer atopical antiviral composition of the present invention and/or a nitricoxide (NO)-releasing active pharmaceutical ingredient to the skin of asubject, thereby treating and/or preventing a viral infection in asubject. A drug delivery system of the present invention may comprise acomposition of the present invention. Example drug delivery systems mayinclude, but are not limited to, substrates, such as a cloth, dressing,membrane, sponge, ring, suppository, and the like, which may be incontact with a composition of the present invention. In someembodiments, a composition of the present invention and/or a nitricoxide (NO)-releasing active pharmaceutical ingredient may be in and/oron a substrate and/or formed into a substrate (e.g., film, suppository,etc.). The substrate may be placed in contact with the skin of a subjectto administer the composition and/or a nitric oxide (NO)-releasingactive pharmaceutical ingredient to the skin of a subject.

Exemplary viral infections include, but are not limited to, a viralinfection caused by cytomegalovirus (CMV), epstein-barr virus, varicellazoster virus (VZV), vaccinia virus, cowpox virus, monkeypox virus,herpes simplex virus (HSV 1+2), herpes zoster, human herpes virus 6(HHV-6), human herpes virus 8 (HHV-8), papillomavirus, molluscumcontagiosum, orf, variola, and/or coxsackie virus. In some embodiments,the viral infection may be caused by a papillomavirus, such as a humanpapillomavirus. The human papillomavirus (HPV) may be HPV type 1, 2, 3,4, 6, 10, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and/or 59. Insome embodiments, the viral infection may be caused by a herpes simplexvirus, such as herpes simplex type 1 and/or herpes simplex type 2. Insome embodiments, the viral infection may infect the skin, includingmucosa, of the subject. In certain embodiments, the virus may be a humanvirus.

According to some embodiments of the present invention, provided hereinare methods of treating and/or preventing virus-related cutaneousconditions. A method of treating and/or preventing a virus-relatedcutaneous condition may comprise administering a topical antiviralcomposition (i.e., a composition of the present invention) to the skinof a subject, thereby treating and/or preventing the virus-relatedcutaneous condition in the subject. Virus-related cutaneous conditionsthat may be treated and/or prevented include, but are not limited to,cutaneous conditions associated with bowenoid papulosis, buffalopox,butcher's wart, condylomata acuminate, cowpox, cytomegalovirus,disseminated herpes zoster, eczema herpeticum (Kaposi's varicelliformeruption), eczema vaccinatum, epidermodysplasia verruciformis, erythemainfectiosum (fifth disease, slapped cheek disease), farmyard pox,generalized vaccinia, genital herpes (herpes genitalis, herpesprogenitalis), Buschke-Löwenstein tumor, hand-foot-and-mouth disease(Coxsackie), Heck's disease (focal epithelial hyperplasia), herpangina,herpes gladiatorum (scrum pox), herpes simplex, herpetickeratoconjunctivitis, herpetic sycosis, herpetic whitlow, humanmonkeypox, human T-lymphotropic virus 1 infection, human tanapox,intrauterine herpes simplex, Kaposi sarcoma, Lipschutz ulcer (ulcusvulvae acutum), Milker's nodule, molluscum contagiosum, neonatal herpessimplex, ophthalmic zoster, orf (contagious pustular dermatosis, ecthymacontagiosum, infectious labial dermatitis, sheep pox), oral floridpapillomatosis, oral hairy leukoplakia (EBV), orolabial herpes (herpeslabialis), progressive vaccinia (vaccinia gangrenosum, vaccinianecrosum), pseudocowpox, recurrent respiratory papillomatosis (laryngealpapillomatosis), sealpox, varicella (chickenpox), variola major(smallpox), verruca plana (flat warts), verruca plantaris (plantarwart), verruca vulgaris (wart), verrucae palmares et plantares, and/orzoster (herpes zoster, shingles).

“Treat,” “treating” or “treatment of” (and grammatical variationsthereof) as used herein refer to any type of treatment that imparts abenefit to a subject and may mean that the severity of the subject'scondition is reduced, at least partially improved or ameliorated and/orthat some alleviation, mitigation or decrease in at least one clinicalsymptom associated with a viral infection is achieved and/or there is adelay in the progression of the viral infection and/or condition. Insome embodiments, the severity of a viral infection (e.g., a viralinfection caused by human papillomavirus) may be reduced in a subjectcompared to the severity of the viral infection in the absence of amethod of the present invention. In certain embodiments, a method of thepresent invention treats a viral infection in a subject, such as a viralinfection that has affected the skin of the subject. In someembodiments, a method of the present invention may treat a viralinfection by eliminating and/or reducing the size and/or appearance ofat least one clinical symptom associated with the viral infection (e.g.,a benign lesion). In some embodiments, a method of the present inventionmay treat a viral infection by eliminating at least one clinical symptomassociated with the viral infection (e.g., a benign lesion) for a givenperiod of time (e.g., 1, 2, 3, 4, 5, or 6 day(s), or 1, 2, 3, 4, or moreweeks, etc.).

In some embodiments, a topical antiviral composition of the presentinvention is administered in a treatment effective amount. A “treatmenteffective” amount as used herein is an amount that is sufficient totreat (as defined herein) a subject. Those skilled in the art willappreciate that the therapeutic effects need not be complete orcurative, as long as some benefit is provided to the subject. In someembodiments, a treatment effective amount of a topical antiviralcomposition of the present invention may be administered and may includeadministering a treatment effective amount of a nitric oxide-releasingactive pharmaceutical ingredient. In some embodiments, a treatmenteffective amount of nitric oxide may be administered and/or applied in amethod of the present invention. In some embodiments, a method of thepresent invention is carried out in a manner such that theadministration of a topical antiviral composition comprising a nitricoxide (NO)-releasing active pharmaceutical ingredient (API) does notproduce systemic effects (e.g., adverse systemic effects) from theadministration of nitric oxide, such as, for example, when thecomposition, NO-releasing API, and/or NO is administered in a treatmenteffective amount. In some embodiments, a method of the present inventionis carried out in a manner such that the administration of a topicalantiviral composition comprising a nitric oxide-releasing activepharmaceutical ingredient produces a local, systemic effect from theadministration of nitric oxide, such as, for example, when thecomposition, NO-releasing API, and/or NO is administered in a treatmenteffective amount.

The terms “prevent,” “preventing” and “prevention” (and grammaticalvariations thereof) refer to avoidance, reduction and/or delay of theonset of a viral infection and/or a clinical symptom associatedtherewith in a subject and/or a reduction in the severity of the onsetof the viral infection and/or clinical symptom relative to what wouldoccur in the absence of a method of the present invention. Theprevention can be complete, e.g., the total absence of the viralinfection and/or clinical symptom. The prevention can also be partial,such that the occurrence of the viral infection and/or clinical symptomin the subject and/or the severity of onset is less than what wouldoccur in the absence of a method of the present invention. In certainembodiments, a method of the present invention prevents a viralinfection in a subject, such as a viral infection that can affect theskin of the subject.

In some embodiments, a topical antiviral composition of the presentinvention is administered in a prevention effective amount. A“prevention effective” amount as used herein is an amount that issufficient to prevent (as defined herein) the viral infection and/orclinical symptom in the subject. Those skilled in the art willappreciate that the level of prevention need not be complete, as long assome benefit is provided to the subject. In some embodiments, aprevention effective amount of a topical antiviral composition of thepresent invention may be administered and may include administering aprevention effective amount of a nitric oxide-releasing activepharmaceutical ingredient. In some embodiments, a prevention effectiveamount of nitric oxide may be administered and/or applied in a method ofthe present invention. In some embodiments, a method of the presentinvention is carried out in a manner such that the administration of atopical antiviral composition comprising a NO-releasing API does notproduce systemic effects (e.g., adverse systemic effects) from theadministration of nitric oxide, such as, for example, when thecomposition, NO-releasing API, and/or NO is administered in a preventioneffective amount. In some embodiments, a method of the present inventionis carried out in a manner such that the administration of a topicalantiviral composition comprising a nitric oxide-releasing activepharmaceutical ingredient produces a local, systemic effect from theadministration of nitric oxide, such as, for example, when thecomposition, NO-releasing API, and/or NO is administered in a preventioneffective amount.

The topical antiviral composition may be topically applied to a subjectusing any method known to those of skill in the art. In someembodiments, the composition may be topically applied to the subject atleast 1, 2, 3, or more times per day. In some embodiments, thecomposition may be topically applied to the subject at least 1, 2, 3, 4,5, 6, 7, 8, or more times per week and/or month. In certain embodiments,the composition may be topically applied to the subject once daily,twice daily, every other day, every third day, once per week, or twiceper week. In some embodiments, the composition may be applied at leastonce daily for an extended period of time (e.g., a week, month, 2months, etc.) and/or until the viral infection and/or clinical symptomassociated therewith has been treated and/or prevented. In someembodiments, the composition may be applied on an as needed basis.

The present invention finds use in both veterinary and medicalapplications. Suitable subjects of the present invention include, butare not limited to avians and mammals. The term “avian” as used hereinincludes, but is not limited to, chickens, ducks, geese, quail, turkeys,pheasants, parrots, parakeets, macaws, cockatiels, canaries, andfinches. The term “mammal” as used herein includes, but is not limitedto, primates (e.g., simians and humans), non-human primates (e.g.,monkeys, baboons, chimpanzees, gorillas), bovines, ovines, caprines,ungulates, porcines, equines, felines, canines, lagomorphs, pinnipeds,rodents (e.g., rats, hamsters, and mice), etc. In some embodiments ofthe present invention, the subject is a mammal and in certainembodiments the subject is a human. Human subjects include both malesand females and subjects of all ages including fetal, neonatal, infant,juvenile, adolescent, adult, and geriatric subjects.

The methods of the present invention may also be carried out on animalsubjects, particularly mammalian subjects such as mice, rats, dogs,cats, livestock and horses for veterinary purposes, and/or for drugscreening and drug development purposes.

In some embodiments, the subject is “in need of” or “in need thereof” amethod of the present invention, for example, the subject is in anat-risk population (e.g. the subject may be at-risk for or moresusceptible to a viral infection), the subject has findings typicallyassociated with a viral infection, and/or the subject is suspected to beor to have been exposed to a virus. In some embodiments, a subject inneed thereof has a viral infection and/or a clinical sign or symptomassociated therewith that may be treated with a method of the presentinvention. The present invention may be particularly suitable forchildren, adolescents, adults, and/or geriatric subjects.

A topical antiviral composition of the present invention may beadministered and/or applied topically to any portion of a subject'sskin, including mucosa. For example, the composition may be topicallyadministered to a subject's hand, finger, foot, toe, arm, leg, trunk,anus, genitals, face, a mucous membrane (including a body cavity), nail,etc. In some embodiments, an antiviral composition of the presentinvention may be topically administered to at least a portion of asubject's hand, finger, foot, and/or toe. In some embodiments, anantiviral composition of the present invention may be topicallyadministered to at least a portion of a subject's anus, genitals, and/ora mucous membrane (e.g., urethra, cervix, and/or vagina). In someembodiments, an antiviral composition of the present invention may betopically administered to at least a portion of a subject's face, lips,and/or a mucous membrane (e.g., nostrils, mouth, tongue, and/orpharynx).

In some embodiments, a method of the present invention may preventand/or reduce the appearance and/or size of a benign lesion. Exemplarybenign lesions include, but are not limited to, a wart (e.g., commonwarts (verruca vulgaris), flat warts, plantar warts, subungual and/orperiungal warts, anal/genital warts, etc.), oral and/or laryngealpapilloma, anogenital mucosal condylomata, focal epithelial hyperplasic,oral florid papillomatosis, condyloma accuminata, papillomata, molluscumcontagiosum, herpetic lesions, orf, and/or cowpow. In some embodiments,the benign lesion may be an external genital wart and/or anal wart(e.g., a perianal wart). In some embodiments, the benign lesion may be anongenital wart. In some embodiments, the benign lesion may be inducedand/or caused by a papillomavirus, such as a human papillomavirus.

A method of the present invention may reduce the appearance and/or sizeof a benign lesion by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 100%compared to the appearance and/or size of a benign lesion prior toadministering of a topical antiviral composition of the presentinvention. The appearance of the benign lesion may be evaluatedvisually, such as, but not limited to, by the subject and/or aphysician. The size of the benign lesion may be determined using methodsknown to those of skill in the art. In some embodiments, a method of thepresent invention may prevent and/or reduce the appearance and/or sizeof a wart.

In certain embodiments, the subject may see a reduction in the sizeand/or appearance of a benign lesion within 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, or more day(s) and/or week(s). In some embodiments, themethod may reduce the size and/or appearance of a benign lesion in theskin of the subject with 12 weeks or less, in some embodiments, within 8weeks or less, and in further embodiments, within 4 weeks or less.

A method of the present invention may reduce the number of benignlesions by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 100% comparedto the number of benign lesions prior to administering of a topicalantiviral composition of the present invention. The number of benignlesions may be evaluated visually, such as, but not limited to, by thesubject and/or a physician. The number of benign lesions may bedetermined using methods known to those of skill in the art. In someembodiments, a method of the present invention may prevent and/or reducethe number of warts.

A method of the present invention may decrease the rate of recurrence ofa benign lesion in a subject by at least about 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,97% or 100% compared to the rate of recurrence of the same type ofbenign lesion in the absence of administering of a topical antiviralcomposition of the present invention. The rate of recurrence may bedetermined using methods known to those of skill in the art. Forexample, after a treatment and/or removal of a benign lesion, the numberof benign lesions may be visually determined after a given period oftime to determine the rate of recurrence. In some embodiments, a methodof the present invention may decrease the rate of recurrence of warts ina subject.

The method may comprise topically administering and/or applying atopical antiviral composition to virally infected skin, includingmucosa, of the subject that comprises a benign lesion. In someembodiments, the virally infected skin comprises a lesion and the methodmay further comprise debriding the lesion prior to administering thetopical composition to the skin of the subject. In other embodiments,the virally infected skin comprises a lesion and the method may notcomprise debriding the lesion prior to administering the topicalcomposition to the skin of the subject. In some embodiments, the lesionmay comprise a wart.

In certain embodiments, a method of the present invention may preventand/or reduce the appearance and/or size of a premalignant lesion and/ora malignant lesion, such as, for example, a tumor. The premalignantlesion and/or malignant lesion may be caused by and/or induced by aviral infection. In some embodiments, a premalignant lesion and/ormalignant lesion may be a premalignant and/or malignant cutaneouslesion. In some embodiments, the premalignant lesion and/or malignantlesion may be due to and/or caused by cancer of the cervix, penis, anus,and/or oral cavity. In some embodiments, the premalignant lesion and/ormalignant lesion may be induced and/or caused by a papillomavirus, suchas a human papillomavirus. In some embodiments, a method of the presentinvention may prevent and/or reduce the appearance and/or size ofcervical intraepithelial neoplasia.

A method of the present invention may reduce the appearance and/or sizeof a premalignant lesion and/or malignant by at least about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 97% or more compared to the appearance and/or size of apremalignant lesion and/or a malignant lesion prior to administering ofa topical antiviral composition of the present invention. The appearanceof the premalignant lesion and/or a malignant lesion may be evaluatedvisually, such as, but not limited to, by the subject and/or aphysician. The size of the premalignant lesion and/or a malignant lesionmay be determined using methods known to those of skill in the art.

A method of the present invention may reduce the number of premalignantlesions and/or malignant lesions by at least about 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 97% or 100% compared to the number of premalignant lesions and/ormalignant lesions prior to administering of a topical antiviralcomposition of the present invention. The number of premalignant lesionsand/or malignant lesions may be evaluated visually, such as, but notlimited to, by the subject and/or a physician. The number ofpremalignant lesions and/or malignant lesions may be determined usingmethods known to those of skill in the art.

A method of the present invention may decrease the rate of recurrence ofa premalignant lesion and/or malignant lesion in a subject by at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 97% or 100% compared to the rate ofrecurrence of the same type of premalignant lesion and/or malignantlesion in the absence of administering of a topical antiviralcomposition of the present invention. The rate of recurrence may bedetermined using methods known to those of skill in the art. Forexample, after a treatment and/or removal of a premalignant lesionand/or malignant lesion, the number of premalignant and/or malignantlesions may be visually determined after a given period of time todetermine the rate of recurrence.

In some embodiments, a method of the present invention may administernitric oxide to the basal layer of a subject's epithelium. A method ofthe present invention may administer a treatment effective and/or aprevention effective amount of nitric oxide to the basal layer of asubject's epithelium. In some embodiments, nitric oxide may beadministered to the basement membrane of a subject's epithelium. Theupper epithelial layers of a subject's skin may not need to be debrided,exfoliated, and/or removed in order for the method to administer nitricoxide to the basal layer and/or basement membrane.

In some embodiments, a method of the present invention may administernitric oxide to the skin of a subject. In some embodiments, a method ofthe present invention may administer nitric oxide in an amountsufficient to induce apoptosis or other cellular damage in virallyinfected cells. In some embodiments, a method of the present inventionmay administer nitric oxide in an amount sufficient to inhibit and/orprevent viral replication. A method of the present invention may reduceviral replication by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%compared to the rate of replication prior to the method of the presentinvention.

In some embodiments, a method of the present invention may treat and/orprevent a viral infection in a subject without cytotoxicity to hostcells or with reduced cytotoxicity to host cells. The method may treatand/or prevent the viral infection in the subject with reduced host cellcytotoxicity compared to a different method for treating the viralinfection, such as, for example, one that does not administer nitricoxide to the skin of a subject or one that uses acidified nitrite. Insome embodiments, a method of the present invention may reduce host cellcytotoxicity by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%compared to a different method for treating the viral infection. Amethod of the present invention may reduce and/or eliminate viralreplication with no or minimal host cell cytotoxicity. For example, themethod may provide a host cell cytotoxicity of about 50% or less (e.g.,about 40%, 30%, 20%, 10%, 5%, or less). Cytotoxicity may be determinedusing methods known to those of skill in the art, such as, for example,a qualitative reading of hematoxylin & eosin (H&E) slides, a lactatedehydrogenase (LDH) assay and/or a 3-(4, 5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. In some embodiments, amethod of the present invention may not cause apoptosis. For example,the method may not cause apoptosis in keratinocyte layers of the skin.

In some embodiments, a method of the present invention may cause cellsin the skin to normalize. The cells may be those that received atherapeutic and/or prophylactic effect from a method of the presentinvention. For example, the cells may be those that were administerednitric oxide according to a method of the present invention. The cellsmay normalize by, for example, returning to a normal growth rate and/ormay complete differentiation. In some embodiments, a method of thepresent invention may reduce the number of actively dividing cellsthroughout the skin layer and may cause cellular division to berestricted to the basal layer of the skin as is the normal physiologicstate. In some embodiments, a method of the present invention may returncells in the skin to a growth rate that does not cause the cells and/orskin to display hyperproliferation, hyperplasia (e.g., benignhyperplasia), and/or dysplasia.

In some embodiments, the virus may cause a thickening in an area of theskin (e.g., the virus may lead to a wart on the skin) and a method ofthe present invention may reduce the thickness of the skin in this area,such as by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,or more and/or may return the thickness of the skin in this area to anormal thickness. In some embodiments, the method may reduce thethickness of a thickened area of skin and/or return the thickness of theskin in the thickened area to a thickness of about 20% or less than thenormal thickness of the skin. For example, an area of normal skin mayhave a thickness of 2 mm and a method of present invention may reducethe thickness of thickened skin in this area to a thickness in a rangeof about 2.4 mm to about 2 mm.

In some embodiments, a method of the present invention may cause cellsin the skin to return to a normal G2 and/or S phase. For example, avirus may cause cells to have a prolonged G2 phase following S phasereentry. In some embodiments, a method of the present invention maydisrupt and/or interfere with a protein involved in viral replication.For example, a method of the present invention may disrupt and/orinterfere with an E7 and/or E6 protein and/or its interactions and/orsignaling. In some embodiments, a method of the present invention mayactivate and/or increase a cellular process that prohibits and/ordecreases viral replication.

In some embodiments, a method of the present invention may reduce theamount of viral DNA. For example, a method of the present invention mayreduce the amount of viral DNA by at least about 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, 99%, or 100% compared to the amount of viral DNA present prior tothe method of the present invention.

According to some embodiments of the present invention, provided is atopical antiviral composition. Exemplary compositions that may be usedas a topical antiviral composition include, but are not limited to,those described in International Application No. PCT/US2014/019536, U.S.Provisional Application No. 61/863,541 filed on Aug. 8, 2013, and U.S.Provisional Application No. 61/868,139 filed on Aug. 21, 2013, thedisclosures of each of which are incorporated herein by reference intheir entirety. The topical antiviral composition may comprise a nitricoxide-releasing active pharmaceutical ingredient (NO-releasing API). Insome embodiments, the topical antiviral composition does not compriseacidified nitrite. “Acidified nitrite”, as used herein, refers to anitric oxide releasing composition where the primary mechanism of nitricoxide release is when a nitrite is reduced, in the presence of an acid,to dinitrogen trioxide, which can dissociate into nitric oxide andnitrous oxide. In further embodiments of the present invention, a methodof the present invention may administer nitric oxide to the skin of asubject without staining the skin of the subject. For example, a methodof the present invention may administer nitric oxide to the skin of asubject without staining the subject's skin yellow, brown, and/or black.

“Nitric oxide releasing active pharmaceutical ingredient” and“NO-releasing API,” as used herein, refer to a compound or othercomposition that provides nitric oxide to the skin of a subject, but isnot gaseous nitric oxide. In some embodiments, the NO-releasing API isalso not acidified nitrite. In some embodiments, the NO-releasing APIincludes a nitric oxide-releasing compound, hereinafter referred to as a“NO-releasing compound.” An NO-releasing compound includes at least oneNO donor, which is a functional group that may release nitric oxideunder certain conditions.

Any suitable NO-releasing compound may be used. In some embodiments, theNO-releasing compound includes a small molecule compound that includesan NO donor group. “Small molecule compound” as used herein is definedas a compound having a molecular weight of less than 500 daltons, andincludes organic and/or inorganic small molecule compounds. In someembodiments, the NO-releasing compound includes a macromolecule thatincludes an NO donor group. A “macromolecule” is defined herein as anycompound that has a molecular weight of 500 daltons or greater. Anysuitable macromolecule may be used, including crosslinked ornon-crosslinked polymers, dendrimers, metallic compounds, organometalliccompounds, inorganic-based compounds, and other macromolecularscaffolds. In some embodiments, the macromolecule has a nominal diameterranging from about 0.1 nm to about 100 μm and may comprise theaggregation of two or more macromolecules, whereby the macromolecularstructure is further modified with an NO donor group.

In some embodiments, the NO-releasing compound includes adiazeniumdiolate functional group as an NO donor. The diazeniumdiolatefunctional group may produce nitric oxide under certain conditions, suchas upon exposure to water. As another example, in some embodiments, theNO-releasing compound includes a nitrosothiol functional group as the NOdonor. The NO donor may produce nitric oxide under certain conditions,such as upon exposure to light. Examples of other NO donor groupsinclude nitrosamine, hydroxyl nitrosamine, hydroxyl amine andhydroxyurea. Any suitable combination of NO donors and/or NO-releasingcompounds may also be used in a second composition as described herein.Additionally, the NO donor may be incorporated into or onto the smallmolecule or macromolecule through covalent and/or non-covalentinteractions.

An NO-releasing macromolecule may be in the form of an NO-releasingparticle, such as those described in U.S. Pat. No. 8,282,967, 8,962,029or 8,956,658, the disclosures of which are incorporated by referenceherein in their entirety. Other non-limiting examples of NO-releasingcompounds include NO-releasing zeolites as described in United StatesPatent Publication Nos. 2006/0269620 or 2010/0331968; NO-releasing metalorganic frameworks (MOFs) as described in United States PatentApplication Publication Nos. 2010/0239512 or 2011/0052650; NO-releasingmulti-donor compounds as described in International Application No.PCT/US2012/052350 entitled “Tunable Nitric Oxide-ReleasingMacromolecules Having Multiple Nitric Oxide Donor Structures”;NO-releasing dendrimers or metal structures as described in U.S.Publication No. 2009/0214618; nitric oxide releasing coatings asdescribed in U.S. Publication No. 2011/0086234; and compounds asdescribed in U.S. Publication No. 2010/0098733. The disclosures of eachof the references in this paragraph are incorporated herein by referencein their entirety. Additionally, NO-releasing macromolecules may befabricated as described in International Application No.PCT/US2012/022048 entitled “Temperature Controlled Sol-GelCo-Condensation” filed Jan. 20, 2012, the disclosure of which isincorporated herein by reference in its entirety.

As an example, in some embodiments of the present invention, a nitricoxide-releasing active pharmaceutical ingredient may include NO-loadedprecipitated silica. The NO-loaded precipitated silica may be formedfrom nitric oxide donor modified silane monomers into a co-condensedsiloxane network. In one embodiment of the present invention, the nitricoxide donor may be an N-diazeniumdiolate. In some embodiments of thepresent invention, the nitric oxide-releasing active pharmaceuticalingredient may comprise, consist essentially of, or consist of aco-condensed siloxane network comprising a diazeniumdiolate (e.g., aN-iazeniumdiolate).

In some embodiments, the nitric oxide donor may be formed from anaminoalkoxysilane by a pre-charging method, and the co-condensedsiloxane network may be synthesized from the condensation of a silanemixture that includes an alkoxysilane and the aminoalkoxysilane to forma nitric oxide donor modified co-condensed siloxane network. As usedherein, the “pre-charging method” means that aminoalkoxysilane is“pretreated” or “precharged” with nitric oxide prior to theco-condensation with alkoxysilane. In some embodiments, the prechargingnitric oxide may be accomplished by chemical methods. In anotherembodiment, the “pre-charging” method may be used to create co-condensedsiloxane networks and materials more densely functionalized withNO-donors. In some embodiments of the present invention, the nitricoxide-releasing active pharmaceutical ingredient may comprise, consistessentially of, or consist of a co-condensed silica network synthesizedfrom the condensation of a silane mixture comprising an alkoxysilane andat least one aminoalkoxysilane having an amine substituted by adiazeniumdiolate (e.g., a N-diazeniumdiolate).

The co-condensed siloxane network may be silica particles with a uniformsize, a collection of silica particles with a variety of size, amorphoussilica, a fumed silica, a nanocrystalline silica, ceramic silica,colloidal silica, a silica coating, a silica film, organically modifiedsilica, mesoporous silica, silica gel, bioactive glass, or any suitableform or state of silica.

In some embodiments, the alkoxysilane is a tetraalkoxysilane having theformula Si(OR)4, wherein R is an alkyl group. The R groups may be thesame or different. In some embodiments the tetraalkoxysilane is selectedas tetramethyl orthosilicate (TMOS) or tetraethyl orthosilicate (TEOS).In some embodiments, the aminoalkoxysilane has the formula:R″—(NH—R′)n—Si(OR)3, wherein R is alkyl, R′ is alkylene, branchedalkylene, or aralkylene, n is 1 or 2, and R″ is selected from the groupconsisting of alkyl, cycloalkyl, aryl, and alkylamine.

In some embodiments, the aminoalkoxysilane may be selected fromN-(6-aminohexyl)aminopropyltrimethoxysilane (AHAP3);N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAP3); (3-trimethoxysilylpropyl)di-ethylenetriamine (DET3);(aminoethylaminomethyl)phenethyltrimethoxysilane (AEMP3);[3-(methylamino)propyl]trimethoxysilane (MAP3);N-butylamino-propyltrimethoxysilane(n-BAP3);t-butylamino-propyltrimethoxysilane(t-BAP3);N-ethylaminoisobutyltrimethoxysilane(EAiB3);N-phenylamino-propyltrimethoxysilane (PAP3); andN-cyclohexylaminopropyltrimethoxysilane (cHAP3).

In some embodiments, the aminoalkoxysilane has the formula: NH[R′—Si(OR)3]2, wherein R is alkyl and R′ is alkylene. In someembodiments, the aminoalkoxysilane may be selected frombis(3-triethoxysilylpropyl)amine, bis-[3-(trimethoxysilyl)propyl]amineand bis-[(3-trimethoxysilyl)propyl]ethylenediamine.

In some embodiments, as described herein above, the aminoalkoxysilane isprecharged for NO-release and the amino group is substituted by adiazeniumdiolate. Therefore, in some embodiments, the aminoalkoxysilanehas the formula: R″—N(NONO—X+)—R′—Si(OR)3, wherein R is alkyl, R′ isalkylene or aralkylene, R″ is alkyl or alkylamine, and X+ is a cationselected from the group consisting of Na+, K+ and Li+.

The composition of the siloxane network, (e.g., amount or the chemicalcomposition of the aminoalkoxysilane) and the nitric oxide chargingconditions (e.g., the solvent and base) may be varied to optimize theamount and duration of nitric oxide release. Thus, in some embodiments,the composition of the silica particles may be modified to regulate thehalf-life of NO release from silica particles.

In another embodiment, the amino group of aminoalkoxysilane issubstituted with a diazeniumdiolate, and the aminoalkoxysilane having aformula of R″—N(NONO—X+)—R′—Si(OR)3, wherein: R is alkyl, R′ is alkyleneor aralkylene, R″ is alkyl or alkylamine, and X+ is a cation selectedfrom the group consisting of Na+ and K+.

In certain embodiments, the NO-releasing API may comprise a co-condensedsilica network comprising diazeniumdiolated aminoethylaminopropyltrimethoxy silane (AEAP3) and tetra methyl orthosilicate (TMOS) and/or aco-condensed silica network comprising diazeniumdiolatedaminoethylaminopropyl trimethoxy silane (AEAP3) and tetraethylorthosilicate (TEOS). In some embodiments, the NO-releasing API maycomprise a co-condensed silica network comprising diazeniumdiolatedmethylaminopropyl trimethoxysilane (MAP3) and tetra methyl orthosilicate(TMOS) and/or a co-condensed silica network comprising diazeniumdiolatedmethylaminopropyl trimethoxysilane (MAP3) and tetraethyl orthosilicate(TEOS).

In some embodiments of the invention, the particle size of aNO-releasing API may be in a range of about 20 nm to about 20 μm or anyrange therein, such as, but not limited to, about 100 nm to about 20 μmor about 1 μm to about 20 μm. The particle size may be tailored tominimize or prevent toxicity and/or penetration through the epidermis(or compromised dermis) and into the blood vessels. In particularembodiments, the particle size is distributed around a mean particlesize of less than 20 μm, or any range therein, and the size may allowthe particle to enter a follicle. In some embodiments, a NO-releasingAPI may have a particle size that is distributed around a mean particlesize of about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or 1 μm. In further embodiments, a NO-releasing API may have aparticle size that is distributed around a mean particle size of lessthan 10 μm, or any range therein, such as, but not limited to about 2 μmto about 10 μm or about 4 μm to about 8 μm. In other embodiments, theparticle size may be distributed around a mean particle size of greaterthan 20 μm, or any range therein, and the size may prevent the particlefrom entering the follicle. In still further embodiments, a mixture ofparticles with mean particle sizes distributed around two or more meanparticle sizes may be provided. A NO-releasing API may be micronized(e.g., ball and/or jet milled). Methods for providing a desired particlesize and/or micronization include, but are not limited to, thosedescribed in U.S. Patent Application Publication No. 2013/0310533, whichis incorporated herein by reference in its entirety.

In some embodiments, a NO-releasing API may be present in a topicalantiviral composition in an amount of about 0.5% to about 25% by weightof the composition. For example, in some embodiments, a NO-releasing APImay be present in a composition of the present invention in an amount ofabout 0.5% to about 20%, about 0.5% to about 5%, about 1% to about 20%,about 1% to about 10%, about 1% to about 8%, about 1% to about 20%,about 5% to about 15%, or about 2% to about 6% by weight of thecomposition. In certain embodiments, a nitric oxide-releasing activepharmaceutical ingredient may be present in a composition of the presentinvention in an amount of about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,23%, 24%, or 25% by weight of the composition.

A composition of the present invention may comprise a NO-releasing APIand may store and/or release nitric oxide in an amount of about 0.05% toabout 10% by weight of the composition, such as, but not limited to,about 0.15% to about 2%, about 0.15% to about 1%, about 0.3% to about1.2%, about 0.15% to about 6%, about 1% to about 10%, about 3% to about6%, or about 1% to about 5% by weight of the composition. In certainembodiments, a composition of the present invention may comprise anitric oxide-releasing active pharmaceutical and may store and/orrelease nitric oxide in an amount of about 0.15%, 0.3%, 0.6%, 0.9%, 1%,1.25%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, 4%,4.25%, 4.5%, 4.75%, 5%, 5.25%, 5.5%, 5.75%, 6%, 6.25%, 6.5%, 6.75%, 7%,7.25%, 7.5%, 7.75%, 8%, 8.25%, 8.5%, 8.75%, 9%, 9.25%, 9.5%, 9.75%, or10% by weight of the composition. The amount of nitric oxide releasedmay be determined using real time in vitro release testing. In someembodiments, nitric oxide release may be determined using achemiluminescent nitric oxide analyzer.

A composition of the present invention may provide and/or allow for anextended period of time of NO release. In some embodiments, acomposition of the present invention may provide and/or allow for acontinuous release of NO for about 1 hour or more, such as, but notlimited to, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more hoursafter administration of the topical composition to a subject. In someembodiments, the composition may provide for a continuous release of NOfor at least about 1, 2, 3, 4, or 5 hours after administration of thetopical composition to a subject.

In some embodiments, a topical antiviral composition of the presentinvention may provide a release rate of about 1 to about 5,000 pmol ofNO/mg/s of the composition at a defined time period after administrationof the composition to a subject. All releases of nitric oxide describedherein, including those described with regard to a time period afteradministration to a subject, are referenced with respect to real time invitro release testing. The in vivo release of nitric oxide (i.e., thenitric oxide release when the topical antiviral composition of thepresent invention is applied to a subject) may vary with the subject towhich the topical antiviral composition is applied. In some embodiments,the in vivo release of nitric oxide may vary depending on the particularembodiment of topical antiviral composition. However, it is believedthat differences in the in vitro release of topical antiviralcompositions according to the present invention will be reflected in therelease of nitric oxide when the topical composition is applied to asubject. Accordingly, for clarity, unless specifically stated that thenitric oxide release is when applied to a subject, references to nitricoxide release with regard to embodiments of the topical antiviralcompositions of the present invention will be with reference to the invitro release of the composition. Time point zero or the initial timepoint of the in vitro release testing may be correlated to the time ofadministration to a subject with all subsequent real-time pointscorresponding to a certain time after administration.

In some embodiments, the composition may release about 1 to about 10,about 1 to about 100, about 100 to about 1000, about 1000 to about4,000, or about 2,500 to about 5,000 pmol of NO/mg of the composition at1 hour, 45, 30, 15, 5, 4, 3, 2, or 1 minute(s) as measured by in vitrorelease. In some embodiments, the composition may release, on average,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, or more pmolof NO/mg of the composition at 24, 20, 15, 10, 5, 4, 3, 2 or 1 hours asmeasured by in vitro release.

In some embodiments, the NO release values provided herein may includethe amount of variation typically associated with the manufacture of theNO-releasing API. For example, variation in the NO release may be seenbetween samples in the same lot and/or different lots. In someembodiments, variation in the NO release between samples in the same lotand/or different lots may be in a range of ±about 0% to about 15% andthis variation may be included in the NO release values describedherein. In some embodiments, variation in the NO release between samplesin the same lot and/or different lots may be in a range of ±about 10% toabout 15% and this variation may be included in the NO release valuesdescribed herein.

In some embodiments, the composition may release about 1 to about 10,about 1 to about 100, about 100 to about 1000, about 1000 to about4,000, or about 2,500 to about 5,000 pmol of NO/mg of the composition at0.5, 1 hour, 45, 30, 15, 5, 4, 3, 2, or 1 minute(s) after administrationof the composition to a subject. In some embodiments, the compositionmay release, on average, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 100, or more pmol of NO/mg of the composition at 24, 20, 15, 10,5, 4, 3, 2 or 1 hours after administration of the composition to asubject.

A topical antiviral composition of the present invention may provide fora continuous release of NO for at least about 0.5, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more hours as measured by in vitro release, and thecomposition may have a release of NO during the continuous release that,on average, is in a range of about 1 to about 500 pmol of NO/mg of thecomposition, such as, but not limited to, about 10 to about 50, about 50to about 200, about 100 to about 500, about 300 to about 500, about 1 toabout 10, or about 1 to about 3 pmol of NO/mg of the composition.

A topical antiviral composition of the present invention may provide fora continuous release of NO for at least about 0.5, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more hours after administration of the composition to asubject, and the composition may have a release of NO during thecontinuous release that, on average, is in a range of about 1 to about500 pmol of NO/mg of the composition, such as, but not limited to, about10 to about 50, about 50 to about 200, about 100 to about 500, about 300to about 500, about 1 to about 10, or about 1 to about 3 pmol of NO/mgof the composition.

In some embodiments of the present invention, a topical antiviralcomposition of the present invention maintains a real time concentrationof NO of greater than 5 pmol of NO/mg for a period of at least 4 hours,a real time concentration of NO of greater than 6 pmol of NO/mg for aperiod of at least 2 hours, and/or a real time concentration of NO ofgreater than 7 pmol of NO/mg for a period of at least 1 hour as measuredby in vitro release. In some embodiments, a topical antiviralcomposition of the present invention may maintain a real timeconcentration of NO of at least about 5 pmol of NO/mg or more (e.g., 10,20, 30, 40, 50, 100, 150, 200, 250, 500, 1000, 2000 pmol of NO/mg of thecomposition or more) for a period of at least 1 hour or more (e.g., 1,1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5 hours ormore) as measured by in vitro release.

In particular embodiments of the present invention, a topical antiviralcomposition of the present invention maintains a real time concentrationof NO in a range of about 5 pmol to about 4000 pmol of NO/mg for aperiod of at least 4 hours, a real time concentration of NO in a rangeof about 6 to about 4000 pmol of NO/mg for a period of at least 2 hours,and/or a real time concentration of NO in a range of about 7 to about4000 pmol of NO/mg for a period of at least 1 hour as measured by invitro release.

In some embodiments of the present invention, a topical antiviralcomposition of the present invention maintains a real time concentrationof NO of greater than 74 pmol of NO/cm² for a period of at least 4hours, a real time concentration of NO of greater than 89 pmol of NO/cm²for a period of at least 2 hours, and/or a real time concentration of NOof greater than 104 pmol of NO/cm² for a period of at least 1 hour asmeasured by in vitro release. In particular embodiments of the presentinvention, a topical antiviral composition of the present inventionmaintains a real time concentration of NO in a range of about 74 pmol ofNO/cm² to about 59,520 pmol of NO/cm² for a period of at least 4 hours,a real time concentration of NO in a range of about 89 to about 59,520pmol of NO/cm² for a period of at least 2 hours, and/or a real timeconcentration of NO in a range of about 104 to 59,520 pmol of NO/cm² fora period of at least 1 hour as measured by in vitro release.

In some embodiments, a topical antiviral composition of the presentinvention may provide for a cumulative release of NO of at least about10 nmol of NO/mg of the composition at 24 hours or less (e.g., 24, 20,15, 10, 5, 4, 2, or 1 hours) after administration of the composition toa subject. The topical antiviral composition may have a cumulativerelease of NO in a range of about 10 to about 50, about 10 to about 100,about 100 to about 1000, about 250 to about 750, about 500 to about 750,about 50 to about 1000, about 100 to about 1500, about 200 to about1000, about 100 to about 500, or about 500 to about 1000 nmol of NO/mgof the composition at 24 hours or less after administration of thecomposition to a subject.

In some embodiments, a topical antiviral composition of the presentinvention provides a cumulative release of NO in a range of about 180nmol of NO/mg to about 1000 nmol of NO/mg in 24 hours afteradministration of the composition to a subject. In some embodiments ofthe present invention, a topical antiviral composition of the presentinvention provides a cumulative release of NO of greater than 180 nmolof NO/mg in 24 hours after administration of the composition to asubject while maintaining a real time concentration of NO of greaterthan 5 pmol of NO/mg for a period of at least 4 hours as measured by invitro release. In some embodiments, a topical antiviral composition ofthe present invention provides a cumulative release of NO in a range ofabout 90 nmol of NO/mg to about 450 nmol of NO/mg in 4 hours afteradministration of the composition to a subject.

In some embodiments, a topical antiviral composition of the presentinvention releases half of the NO released from the composition in about9 minutes or longer based on a total NO release determined at 24 hoursmeasured by in vitro release. In some embodiments, the topical antiviralcomposition may release half of the NO released from the composition inabout 10, 20, 30, 40, 50, or 60 minutes, or 2, 3, 4, 5, 6, 7, or 8 hoursor more based on a total NO release determined at 24 hours measured byin vitro release. In some embodiments, a topical antiviral compositionof the present invention releases half of the NO released from thecomposition in a range of about 9 minutes to about 8 hours based on atotal NO release determined at 24 hours measured by in vitro release.

In some embodiments, a topical antiviral composition of the presentinvention provides a maximum concentration (Cmax) of NO released ofgreater than 160 pmol of NO/mg based on a total NO release determined at24 hours measured by in vitro release. In some embodiments, the topicalantiviral composition of the present invention may provide a Cmax of NOreleased of about 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000,1500, 2000, 2500, 3000, 3500 pmol of NO/mg or more based on a total NOrelease determined at 24 hours measured by in vitro release. In someembodiments, a topical antiviral composition of the present inventionprovides a maximum concentration of NO released in a range of about 160pmol of NO/mg to about 3500 pmol of NO/mg. In some embodiments, atopical antiviral composition of the present invention provides amaximum concentration of NO released of greater than 160 pmol of NO/mgand releases half of the NO released from the composition in about 9minutes or longer based on a total NO release determined at 24 hoursmeasured by in vitro release.

In particular embodiments of the present invention, a topical antiviralcomposition provides a maximum concentration of NO released of at leastabout 3000 pmol of NO/mg, releases at least about 900 nmol of NO/mg in24 hours, and/or releases half of the NO release in about 9 minutesbased on a total NO release determined at 24 hours measured by in vitrorelease. In particular embodiments of the present invention, a topicalantiviral composition provides a maximum concentration of NO released ofat least about 13 pmol of NO/mg, releases at least about 300 nmol ofNO/mg in 24 hours and/or releases half of the NO release in about 420minutes based on a total NO release determined at 24 hours measured byin vitro release. In further embodiments, a topical antiviralcomposition has a real time concentration of NO of at least 5 pmol ofNO/mg at 4 hours as measured by in vitro release. In particularembodiments of the present invention, a topical antiviral compositionprovides a maximum concentration of NO released in a range of about 12pmol of NO/mg to about 3200 pmol of NO/mg, a real time concentration ofNO of at least 5 pmol of NO/mg at 4 hours, releases NO in a range ofabout 300 nmol of NO/mg to about 1000 nmol of NO/mg in 24 hours, and/orreleases half of the NO release in a range of about 9 minutes to about420 minutes based on a total NO release determined at 24 hours measuredby in vitro release.

In some embodiments, a topical antiviral composition of the presentinvention may maintain a real time concentration of NO of at least about70 pmol of NO/cm² or more (e.g., 75, 100, 150, 200, 250, 500, 1000,2000, 3000, 4000, 5000, 6000, 7000 pmol of NO/cm² or more) fora periodof at least 0.5 hours or more (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5 hours or more) after administration of thecomposition to a subject, as measured by in vitro release. In someembodiments of the present invention, a topical antiviral composition ofthe present invention maintains a real time concentration of NO ofgreater than 70 pmol of NO/cm² for a period of at least 4 hours asmeasured by in vitro release.

In some embodiments, a topical antiviral composition of the presentinvention may provide for a cumulative release of NO of at least about4500 nmol of NO/cm² (e.g., 5000, 6000, 7000, 8000, 9000, 10000, 11000,12000, 13000, 14000 nmol of NO/cm² or more) at 24 hours or less (e.g.,24, 20, 15, 10, 5, 4, 2, or 1 hours) after administration of thecomposition to a subject. In some embodiments, a topical antiviralcomposition of the present invention provides a cumulative release of NOin a range of about 4500 nmol of NO/cm² to about 14000 nmol of NO/cm² in24 hours after administration of the composition to a subject. In someembodiments of the present invention, a topical antiviral composition ofthe present invention provides a cumulative release of NO of greaterthan 4500 nmol of NO/cm² in 24 hours after administration of thecomposition to a subject while maintaining a real time concentration ofNO of greater than 70 pmol of NO/cm² for a period of at least 4 hours asmeasured by in vitro release. In some embodiments, a topical antiviralcomposition of the present invention provides a cumulative release of NOin a range of about 1300 nmol of NO/cm² to about 14000 nmol of NO/cm² in4 hours after administration of the composition to a subject.

In some embodiments, a topical antiviral composition of the presentinvention may have a real time concentration of NO of at least about 10pmol of NO/cm² or more (e.g., 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000 pmol of NO/cm² or more) at 0.5hours or more (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, 8, 8.5 hours or more) after administration of the composition to asubject. In some embodiments, a topical antiviral composition of thepresent invention provides a real time concentration of NO of at least100 pmol of NO/cm² at 0.5 hours, at least 50 pmol of NO/cm² at 1 hour,at least 40 pmol of NO/cm² at 2 hours, at least 25 pmol of NO/cm² at 3hours, and/or at least 20 pmol of NO/cm² at 4 hours, each as measured byin vitro release. In some embodiments, a topical antiviral compositionof the present invention provides a real time concentration of NO of atleast 130 pmol of NO/cm² at 0.5 hours, at least 115 pmol of NO/cm² at 1hour, at least 90 pmol of NO/cm² at 2 hours, at least 90 pmol of NO/cm²at 3 hours, and/or at least 80 pmol of NO/cm² at 4 hours, each asmeasured by in vitro release.

In some embodiments, a topical antiviral composition of the presentinvention provides a real time concentration of NO of at least 800 pmolof NO/cm² at 0.5 hours, at least 500 pmol of NO/cm² at 1 hour, at least200 pmol of NO/cm² at 2 hours, at least 100 pmol of NO/cm² at 3 hours,and/or at least 50 pmol of NO/cm² at 4 hours, each as measured by invitro release.

In some embodiments, a topical antiviral composition of the presentinvention provides a maximum concentration of NO released of greaterthan 2400 pmol of NO/cm². In some embodiments, a topical antiviralcomposition of the present invention provides a maximum concentration ofNO released in a range of about 2400 pmol of NO/cm² to about 47000 pmolof NO/cm². In some embodiments, a topical antiviral composition of thepresent invention provides a maximum concentration of NO released ofgreater than 2400 pmol of NO/cm² and releases half of the NO releasedfrom the composition in 10 minutes or longer based on a total NO releasedetermined at 24 hours measured by in vitro release.

In particular embodiments of the present invention, a topical antiviralcomposition provides a maximum concentration of NO released of at leastabout 47000 pmol of NO/cm², releases at least about 13000 nmol of NO/cm²in 24 hours and/or releases half of the NO release in about 9 minutesbased on a total NO release determined at 24 hours measured by in vitrorelease. In particular embodiments of the present invention, a topicalantiviral composition provides a maximum concentration of NO released ofat least about 190 pmol of NO/cm², releases at least about 4600 nmol ofNO/cm² in 24 hours and/or releases half of the NO in about 420 minutesbased on a total NO release determined at 24 hours measured by in vitrorelease. In further embodiments, a topical antiviral composition has areal time concentration of NO of at least 70 pmol of NO/cm² at 4 hoursas measured by in vitro release. In particular embodiments of thepresent invention, a topical antiviral composition provides a maximumconcentration of NO release in a range of about 190 pmol of NO/cm² toabout 47000 pmol of NO/cm², a real time concentration of NO of at least70 pmol of NO/cm² at 4 hours, releases NO in a range of about 4600 nmolof NO/cm² to about 47000 nmol of NO/cm² in 24 hours and/or releases halfof the NO release in a range of about 9 minutes to about 420 minutesbased on a total NO release determined at 24 hours measured by in vitrorelease.

The efficacy of the nitric oxide releasing product may be related, notonly to the amount of nitric oxide release, but to the rate at which itis released. Accordingly, products with an average release rate overabout 5 minutes and in some embodiments, 4.7 minutes, of 600 nmol NO/mghour^(0.5) or greater, 900 nmol NO/mg hour^(0.5) or greater, 2500 nmolNO/mg hour^(0.5) or greater, 4000 nmol NO/mg hour^(0.5) or greater or4500 nmol NO/mg hour^(0.5) or greater measured by in vitro release maybe utilized according to certain embodiments of the present invention.

The pH of a composition of the present invention may be in a range ofabout 3 to about 11. In some embodiments, the pH of the composition maybe in a range of about 3 to about 5, about 3.5 to about 4.5, about 5 toabout 7, about 5.5 to about 6.5, about 3.5 to about 6.5, about 6 toabout 10, about 6 to about 7, about 7 to about 10, about 7 to about 9,about 7 to about 8, about 7.5 to about 8, or about 8 to about 9. Incertain embodiments, the pH of the composition may be about 3, 4, 5, 6,7, 8, 9, 10, or 11. The pH of the composition may be determined prior toadministration to a subject and/or may be determined once applied to theskin of the subject. In some embodiments, where a composition of thepresent invention comprises two or more parts and/or phases, the pH maybe determined upon combination and/or mixing of the two or more partsand/or phases prior to and/or after administration to the skin of thesubject. In certain embodiments, the pH of the composition is measuredprior to administration to the skin of the subject, but after combiningall parts and/or phases of the composition.

The pH may be measured using known methods. The pH of a composition ofthe present invention may be determined once a steady state pH isachieved prior to and/or after administration and/or after combinationof a first part and second part of the composition. Alternatively or inaddition, the pH of a composition of the present invention may bedetermined after a defined period of time, such as, but not limited to,after about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes ormore. In some embodiments, the pH of a composition of the presentinvention may be measured in vitro. Alternatively or in addition, the pHof a composition of the present invention may be measured afteradministration to a subject, such as, for example, a skin surface pH maybe measured after administration of a composition of the presentinvention to the skin of a subject.

A composition of the present invention may comprise at least two parts.The at least two parts may be combined prior to, during, and/or afteradministration to a subject to form an antiviral composition of thepresent invention. In some embodiments, a composition of the presentinvention comprises a first part comprising a first composition and asecond part comprising a second composition.

In some embodiments, the first and second composition may be combined bymixing, stirring, blending, dispersing, milling, homogenizing, applyingto same area or region, and the like. In some embodiments, the first andsecond composition may be mixed and/or blended prior to, during, and/orafter administration to the skin of a subject. In some embodiments, thefirst composition and second composition may be combined by applying oneor more layers of the second composition onto a subject and thenapplying one or more layers of the first composition onto a subject orvice versa to form a topical antiviral composition of the presentinvention.

The second composition may comprise a NO-releasing API. In someembodiments, a composition of the present invention may comprise a firstpart comprising a first composition that may be in the form of ahydrogel. “Hydrogel,” as used herein, refers to a hydrophilic gelcomprising a gel matrix and water. In some embodiments, the firstcomposition may comprise at least one polyhydric alcohol, at least oneviscosity increasing agent, and water. In some embodiments, the firstcomposition may comprise at least one polyhydric alcohol, at least oneviscosity increasing agent, water, at least one additional solvent(i.e., at least one solvent in addition to water), a buffer and/orbuffering agent, an emollient, and optionally a preservative.

Exemplary polyhydric alcohols that may be present in a first compositioninclude, but are not limited to, glycerol, propylene glycol,polyethylene glycol, polypropylene glycol, triethylene glycol, neopentalglycols, butylene glycol, polyethylene glycol, sorbitol, arabitol,erythritol, HSH, isomalt, lactitol maltitol, mannitol, xylitol,threitol, ribitol, galactitol, fucitol, iditol, inositol, volemitol, andany combination thereof. In some embodiments, the first compositioncomprises glycerol, such as, but not limited to, anhydrous glycerol.

A polyhydric alcohol may be present in a first composition in an amountof about 1% to about 30% by weight of the first composition or any rangeand/or individual value therein, such as, but not limited to, about 1%to about 20%, about 1% to about 10%, about 5% to about 10%, or about 5%to about 15% by weight of the first composition. In certain embodiments,a polyhydric alcohol may be present in the first composition in anamount of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, or 30% by weight of the first composition or any range and/orindividual value therein.

Exemplary viscosity increasing agents that may be present in a firstcomposition include, but are not limited to, a carboxypolymethylene; apolyacrylic polymer such as polyacrylic acid, a polyacrylate polymer, across-linked polyacrylate polymer, a cross-linked polyacrylic acid, andmixtures thereof a cellulose ether such as hydroxyalkyl cellulosepolymers such as hydroxypropyl methyl cellulose (HPMC), hydroxypropylcellulose, hyrdoxyethyl cellulose, methyl cellulose, carboxymethylcellulose, and mixtures thereof a methacrylate; a polyvinylpyrollidone;cross-linked polyvinyl pyrrolidone; polyvinylpyrrolidone-vinyl acetatecopolymer; polyvinylalcohol; polyethylene oxide; polyethylene glycol;polyvinylalkyl ether-maleic acid copolymer; a carboxy vinyl polymer; apolysaccharide; a gum such as sodium alginate, carrageenan, xantham gum,gum acacia, arabic gum, guar gum, pullulan, agar, chitin, chitosan,pectin, karaya gum, zein, hordein, gliadin, locust bean gum,tragacantha, and mixtures thereof a protein such as collagen, wheyprotein isolate, casein, milk protein, soy protein, gelatin, andmixtures thereof; a starch such as maltodextrin, amylose, high amylosestarch, corn starch, potato starch, rice starch, tapioca starch, peastarch, sweet potato starch, barley starch, wheat starch, waxy cornstarch, modified starch (e.g. hydroxypropylated high amylose starch),dextrin, levan, elsinan, gluten, and mixtures thereof; bentonite;calcium stearate; ceratonia; colloidal silicon dioxide; dextrin;hypromellose; polycarbophil; kaolin; saponite; sorbitan esters; sucrose;sesame oil; tragacanth; potassium alginate; povidone; sodium starchglycolate; phospholipids; and any combination thereof.

In some embodiments, a first composition may comprise acarboxypolymethylene, such as, but not limited to, those commerciallyavailable from Lubrizol Corporation of Wickliffe, Ohio under the tradename Carbopol®. Exemplary Carbopol® polymers that may be present in afirst composition include, but are not limited to, Carbopol® 974P NFpolymer, such as Type A, Type B and/or Type C Homopolymers; Carbopol®Ultrez 10, 20, 21 NF polymer; Carbopol® 971P NF polymer; Carbopol® 980Homopolymer Type C polymer, Carbopol® 980 NF polymer, Carpobol® 980Ppolymer, Carbopol® ETD 2020 NF polymer, Carbopol® 71 G NF polymer,Carbopol® 981P NF polymer, Carbopol® 970P NF polymer, Carbopol® 981P NFpolymer, Carbopol® 5984P NF polymer, Carbopol® 934P NF polymer,Carbopol® 940P NF polymer, Carbopol® 941P NF polymer, Carbopol® 13242 NFpolymer, Carbopol® AA-1 USP NF polymer, Carbopol® TR1 NF polymer,Carbopol® TR2 NF polymer, Lubrizol Aqua CC polymer and SF-2 polymer, andany combination thereof

In some embodiments, a first composition may comprise a cellulose, suchas, but not limited to, a carboxymethyl cellulose or a salt thereof. Insome embodiments, a first composition may comprise carboxymethylcellulose sodium.

In some embodiments, a viscosity increasing agent present in the firstcomposition may be a polymer comprising acidic groups, such as, but notlimited to, carboxylic acid groups. The acidic groups of the polymer maybe partially neutralized in the first composition. In certainembodiments, a viscosity increasing agent present in the firstcomposition may be a carboxypolymethylene. In some embodiments, acarboxypolymethylene present in the first composition may be partiallyneutralized. A first composition may comprise a carboxypolymethylene andhave a pH of about 3 to about 7, about 3.5 to about 6.5, about 3.5 toabout 6, or about 4 to about 6. In certain embodiments, a firstcomposition may comprise a carboxypolymethylene and have a pH of about3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7.

A viscosity increasing agent may be present in the first composition. Insome embodiments, a composition of the present invention may comprise atleast two viscosity increasing agents that may be the same or different.In some embodiments, a first viscosity increasing agent may be presentin a first composition in an amount of about 0.01% to about 5% by weightof the first composition or any range and/or individual value therein,such as, but not limited to, about 0.05% to about 3%, about 1% to about5%, about 1% to about 3%, or about 0.1% to about 1.5% by weight of thefirst composition. In certain embodiments, a first viscosity increasingagent is present in a first composition in an amount of about 0.01%,0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%,4%, 4.5%, or 5% by weight of the first composition or any range and/orindividual value therein.

Water may be present in the first composition in an amount of about 55%to about 99% by weight of the first composition or any range and/orindividual value therein, such as, but not limited to, about 55% toabout 75%, about 60% to about 95%, about 60% to about 80%, about 75% toabout 95%, or about 80% to about 90% by weight of the first composition.In certain embodiments, water is present in a first composition in anamount of about 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% by weight of the first composition orany range and/or individual value therein.

In some embodiments, one or more solvent(s) in addition to water may bepresent in a first composition. For example, the first composition maycomprise water and 1, 2, 3, 4, 5, or more additional solvents. The oneor more solvent(s) in addition to water may each be present in the firstcomposition in an amount of about 0.5% to about 20% by weight of thefirst composition or any range and/or individual value therein, such as,but not limited to, about 1% to about 15%, about 1% to about 10%, about5% to about 15%, or about 1% to about 5% by weight of the firstcomposition. In certain embodiments, the one or more solvent(s) inaddition to water may each be present in a first composition in anamount of about 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% by weight of thefirst composition or any range and/or individual value therein. Examplesolvents in addition to water that may be present in a first compositioninclude, but are not limited to an alcohol, such as, for example,isopropyl alcohol or ethanol.

In some embodiments, a first composition comprises, consists essentiallyof, or consists of at least one polyhydric alcohol present in an amountof about 1% to about 30% by weight of the first composition, at leastone viscosity increasing agent present in an amount of about 0.1% toabout 5% by weight of the first composition, and water present in anamount of about 55% to about 99% by weight of the first composition. Incertain embodiments, the viscosity increasing agent may be acarboxypolymethylene or a carboxymethyl cellulose or a salt thereof. Thefirst composition may be a hydrogel.

A first composition may comprise a preservative. A preservative may bepresent in a first composition in an amount of about 0.01% to about 1%by weight of the first composition or any range and/or individual valuetherein, such as, but not limited to, about 0.01% to about 0.1%, about0.05% to about 0.5%, about 0.05% to about 1%, or about 0.1% to about 1%by weight of the first composition. In certain embodiments, apreservative is present in a first composition in an amount of about0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%,0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% by weight of thefirst composition or any range and/or individual value therein.Exemplary preservatives that may be present in a first compositioninclude, but are not limited to, sorbic acid, benzoic acid,methyl-paraben, propyl-paraben, methylchloroisothiazolinone,metholisothiazolinone, diazolidinyl urea, chlorobutanol, triclosan,benzethonium chloride, p-hydroxybenzoate, chlorhexidine, digluconate,hexadecyltrimethyl ammonium bromide, alcohols, benzalkonium chloride,boric acid, bronopol, butylparaben, butylene calcium acetate, calciumchloride, calcium lactate, carbon dioxide, cationic, and bentonite,cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol,chlorocresol, chloroxylenol, citric acid monohydrate, cresol, dimethylether, ethylparaben, glycerin, hexetidine, imidurea, isopropyl alcohol,lactic acid, monothioglycerol, pentetic acid, phenol, phenoxyethanol,phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate,phenylmercuric nitrate, potassium benzoate, potassium metabisulfite,potassium sorbate, propionic acid, propyl gallate, propylene glycol,sodium acetate, sodium benzoate, sodium borate, sodium lactate, sodiumsulfite, sodium propionate, sodium metabisulfite, xylitol, sulphurdioxide, carbon dioxide, and any combination thereof.

A first composition may comprise a neutralizing agent. A neutralizingagent may be present in a first composition in an amount sufficient toprovide a desired pH, such as, but not limited to, a pH of about 3 toabout 11, or any range and/or individual value therein, such as, but notlimited to, about 3 to about 8, about 4 to about 7, or about 6 to about7, or about 6 to

In some embodiments, a neutralizing agent may be present in a firstcomposition in an amount sufficient to provide the first compositionwith a pH in a range of about 3 to about 8.

In certain embodiments, a neutralizing agent may be present in a firstcomposition in an amount sufficient to provide a composition of thepresent invention with a desired pH upon combination of the firstcomposition and a second part (e.g., a second composition) and/or uponadministration of the first composition and/or the compositioncomprising the first composition and the second part to the skin of asubject. A neutralizing agent may be present in a first composition inan amount sufficient to provide a composition of the present invention(e.g., a composition comprising the first composition and a secondcomposition) with a desired pH, such as, but not limited to, a pH ofabout 3 to about 11, or any range and/or individual value therein.

In some embodiments, a neutralizing agent adjusts the pH of the firstcomposition and/or composition of the present invention. In certainembodiments of the present invention, a neutralizing agent is present ina first composition in an amount sufficient for the first composition tohave a pH of about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8 or anyrange and/or individual value therein. In some embodiments of thepresent invention, a neutralizing agent is present in a composition ofthe present invention in an amount sufficient for the composition tohave a pH of about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10, 10.5, or 11, or any range and/or individual value therein.

Exemplary neutralizing agents that may be present in a first compositioninclude, but are not limited to, bases such as sodium hydroxide,potassium hydroxide, and mixtures thereof; acids such as hydrochloricacid, citric acid, lactic acid, glycolic acid, acetic acid, and mixturesthereof; sodium carbonate; trolamine; tromethamine; aminomethylpropanol; triisopropanolamine; aminomethyl propanol; tetrahydroxypropylethylenediamine; tetrasodium EDTA; suttocide A; and any combinationthereof

A neutralizing agent may be present in a first composition in an amountof about 0.01% to about 1% by weight of the first composition or anyrange and/or individual value therein, such as, but not limited to,about 0.01% to about 0.1%, about 0.05% to about 1%, or about 0.1% toabout 1% by weight of the first composition. In certain embodiments, aneutralizing agent may be present in a first composition in an amount ofabout 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%,0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% by weight ofthe first composition or any range and/or individual value therein.

A first composition may be unbuffered or buffered. In some embodiments,a first composition may be unbuffered. In other embodiments, a firstcomposition may be buffered. Exemplary buffers that may be present in afirst composition include, but are not limited to, acetic acid/acetatebuffers; hydrochloric acid/citrate buffers; citro-phosphate buffers;phosphate buffers; citric acid/citrate buffers; lactic acid buffers;tartaric acid buffers; malic acid buffers; glycine/HCl buffers; salinebuffers such as phosphate buffered saline (PBS), Tris-buffered saline(TBS), Tris-HCl, NaCl, Tween buffered saline (TNT), phosphate bufferedsaline, Triton X-100 (PBT) and mixtures thereof cacodylate buffers;barbital buffers; tris buffers; and any combination thereof. In someembodiments, the buffer may be a phosphate buffer, such as, for example,a potassium phosphate dibasic and/or potassium phosphate monobasicbuffer.

In some embodiments, a buffer may be present in a first composition inan amount of about 0.01% to about 20% by weight of the first compositionor any range and/or individual value therein, such as, but not limitedto, about 0.1% to about 20%, about 1% to about 15%, about 5% to about20%, about 10% to about 20%, or about 1% to about 10% by weight of thefirst composition. In certain embodiments, a buffer is present in afirst composition in an amount of about 0.01%, 0.02%, 0.03%, 0.04%,0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%,0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% by weight of the firstcomposition or any range and/or individual value therein.

In certain embodiments, a first composition may comprise a bufferingagent. Exemplary buffering agents include, but are not limited to,citric acid, acetic acid, lactic acid, boric acid, succinic acid, malicacid, and any combination thereof. A buffering agent may be present in afirst composition in an amount of about 0.01% to about 4% by weight ofthe first composition or any range and/or individual value therein, suchas, but not limited to, about 0.01% to about 0.1%, about 0.05% to about1%, about 0.1% to about 0.5%, about 1% to about 3%, or about 0.1% toabout 2% by weight of the first composition. In certain embodiments, abuffering agent is present in a first composition in an amount of about0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%,0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.25%, 1.5%, 1.75%,2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, or 4% by weight of thefirst composition or any range and/or individual value therein.

In some embodiments, a buffer and/or buffering agent is present in afirst composition in an amount sufficient for the first composition tohave a pH of about 3 to about 8 or any range and/or individual valuetherein, such as, but not limited to, about 3 to about 6, about 3 toabout 5, about 4 to about 7, about 5 to about 7, or about 6 to about 7.In certain embodiments of the present invention, a buffer and/orbuffering agent may be present in a first composition in an amountsufficient for the first composition to have a pH of about 3, 3.5, 4,4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8, or any range and/or individual valuetherein.

In some embodiments, a buffer and/or buffering agent may be present in afirst composition in an amount sufficient to provide a desired pH for acomposition of the present invention comprising the first compositionand a second part (e.g., a second composition). For example, acomposition of the present invention may comprise a second compositionand a first composition comprising a buffer and/or buffering agent,wherein the buffer and/or buffering agent is present in an amountsufficient to provide the composition with a pH of about 3 to about 11,such as, but not limited to, about 3 to about 8, about 7 to about 11,about 8 to about 10, about 3 to about 5, about 4 to about 7, about 5 toabout 7, or about 6 to about 7. In certain embodiments of the presentinvention, a buffer and/or buffering agent may be present in a firstcomposition in an amount sufficient for the composition of the presentinvention to have a pH of about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, 9, 9.5, 10, 10.5, or 11, or any range and/or individual valuetherein. In some embodiments, the buffer and/or buffering agent may bepresent in a first composition in an amount sufficient to provide adesired pH upon administration of a composition of the present inventioncomprising the first composition and a second part to the skin of asubject.

In some embodiments, a buffer, buffering agent, and/or neutralizingagent may be present in a first composition in an amount sufficient toprovide a composition of the present invention and/or a firstcomposition with a desired pH.

In some embodiments, an emollient may be provided in a firstcomposition, such as, but not limited to, silicones, such as, forexample, cyclomethicone, dimethicone, simethicone, C26-28 alkyldimethicone, C26-28 alkyl methicone, polyphenylsisquioxane, trimethylsiloxysilicate and crosspolymers of cyclopentasiloxane anddimethicone/vinyltrimethylsiloxysilicate, and blends thereof. In someembodiments, a first composition may comprise cyclomethicone. Anemollient may be present in the first composition in an amount of about0.5% to about 10% by weight of the first composition or any range and/orindividual value therein, such as, but not limited to, about 1% to about5%, about 0.5% to about 4%, about 1% to about 10%, or about 2% to about8% by weight of the first composition. In certain embodiments, anemollient may be present in the first composition in an amount of about0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% by weight of the firstcomposition or any range and/or individual value therein.

In certain embodiments, a first composition may comprise at least onepolyhydric alcohol present in an amount of about 1% to about 30% byweight of the first composition, at least one viscosity increasing agentpresent in an amount of about 0.01% to about 5% by weight of the firstcomposition, water present in an amount of about 55% to about 99% byweight of the first composition, and optionally at least onepreservative in an amount of about 0.01% to about 1% by weight of thefirst composition. The first composition may have a pH in a range ofabout 3 to about 8, about 3 to about 6, or about 6 to about 8 and may bebuffered. The first composition may be a hydrogel.

In some embodiments, a first composition may comprise, consistessentially of, or consist of a polyhydric alcohol in an amount of about1% to about 15% by weight of the first composition, a viscosityincreasing agent in an amount of about 0.1% to about 5% by weight of thefirst composition, water in an amount of about 55% to about 85% byweight of the first composition, optionally a buffer in an amount ofabout 0.1% to about 20%, optionally a buffering agent in an amount ofabout 0.001% to about 2% by weight of the first composition, optionallya preservative in an amount of about 0.001% to about 1% by weight of thefirst composition, and optionally a neutralizing agent in an amount ofabout 0.001% to about 1% by weight of the first composition. The firstcomposition may have a pH in a range of about 3 to about 5 or about 5 toabout 7. In certain embodiments, the viscosity increasing agent presentin the first composition may be a carboxypolymethylene or carboxymethylcellulose or a salt thereof. In some embodiments, the first compositionmay be cosmetically elegant. The first composition may be a hydrogel.

In some embodiments, a first composition may comprise, consistessentially of, or consist of a polyhydric alcohol in an amount of about1% to about 30% by weight of the first composition, a viscosityincreasing agent in an amount of about 0.01% to about 5% by weight ofthe first composition, water in an amount of about 55% to about 99% byweight of the first composition, optionally at least one additionalsolvent (e.g., an alcohol) in an amount of about 0.5% to about 20% byweight of the first composition, optionally an emollient in an amount ofabout 0.5% to about 10% by weight of the first composition, optionally abuffer in an amount of about 0.01% to about 20% by weight of the firstcomposition, optionally a preservative in an amount of about 0.001% toabout 1% by weight of the first composition, and optionally aneutralizing agent in an amount of about 0.001% to about 1% by weight ofthe first composition. In some embodiments, the at least one solventcomprises an alcohol and the first composition comprises a buffer, anemollient, and a preservative. The first composition may have a pH in arange of about 3 to about 5 or about 5 to about 7. In some embodiments,the pH may be about 4.5. In certain embodiments, the viscosityincreasing agent present in the first composition may becarboxymethylcellulose or a salt thereof. In some embodiments, the firstcomposition may be cosmetically elegant. The first composition may be ahydrogel.

A composition of the present invention may comprise an activepharmaceutical ingredient (API). Except for acidified nitrite, anysuitable API or combinations of APIs may be included in a composition ofthe present invention. In some embodiments, the API may be any suitableAPI that provides the nitric oxide release as described herein. Examplesof APIs include, but are not limited to, antimicrobial agents, anti-acneagents, anti-inflammatory agents, analgesic agents, anesthetic agents,antihistamine agents, antiseptic agents, immunosuppressants,antihemorrhagic agents, vasodilators, wound healing agents, anti-biofilmagents, and any combination thereof. Exemplary APIs include, but are notlimited to, those described in International Application Publication No.WO 2013/006608, which is incorporated herein by reference in itsentirety.

In some embodiments, a first composition may not comprise an API. Incertain embodiments, a first composition does not contain a nitric oxide(NO) releasing API. In some embodiments, a first composition maycomprise at least one API, but the first composition may not comprise anNO-releasing API. In some embodiments, a first composition comprises anAPI (e.g., a moisture sensitive API) and a second composition comprisesa second API, such as, for example, a NO-releasing API.

In some embodiments, the second composition may be an anhydrouscomposition. “Anhydrous,” as used herein, means that there is no directaddition of water to the second composition when it is being prepared.However, those skilled in the art will recognize that water may bephysically and/or chemically absorbed by the second composition and/orby one or more ingredients in the second composition at any time duringthe preparation, storage, and/or use of the second composition (i.e.,indirect addition of water to the second composition). In someembodiments, the term “anhydrous” means that the second composition hasa water content of less than 5% by weight of the second composition orany range and/or individual value therein. A second composition may havea water content of less than 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, or 0.5%,or any range therein, by weight of the second composition. Water contentmay be measured by methods known to those of skill in the art, such as,but not limited to, Karl Fischer titration. In certain embodiments, uponcontact with a second composition, a composition of the presentinvention adds water to the second composition and/or the secondcomposition absorbs water from a composition of the present invention.

Exemplary second compositions that may be used and/or placed in contactwith a first composition include, but are not limited to, thosedescribed in International Application Publication No. WO 2013/006608,which is incorporated herein by reference in its entirety. An exemplarysecond composition that may be used and/or placed in contact with afirst composition to form a topical antiviral composition of the presentinvention may comprise an anhydrous composition comprising at least oneviscosity increasing agent present in the second composition in anamount of about 0.1% to about 30% by weight of the second composition,at least one organic solvent present in the second composition in anamount of about 50% to about 90 by weight of the second composition, andat least one humectant present in the second composition in an amount ofabout 2% to about 20% by weight of the second composition. The secondcomposition may further comprise at least one water repelling agent,also referred to as a water repellant.

Exemplary viscosity increasing agents for the second compositioninclude, but are not limited to, co-polymers of carboxymethylcelluloseand acrylic acid, N-vinylpyrrolidone, polyalkylene glycols (e.g.,poly(ethylene glycol)), polyalkylene oxides (e.g., polyethylene oxide),polyvinyl alcohols, polyvinylpyrrolidone, polysiloxanes, poly(vinylacetates), cellulose, derivatized celluloses, alginates, copolymersthereof and blends thereof. A specific example of a viscosity agent forthe second composition is a hydroxypropylcellulose, such as Klucel®hydroxypropylcellulose (e.g., Klucel® MF Pharm grade). A viscosityincreasing agent may be present in the second composition in an amountof about 0.1% to about 30% by weight of the second composition or anyrange and/or individual value therein, such as, but not limited to,about 0.5% to about 20%, about 0.1% to about 2%, about 0.5% to about 5%,about 1% to about 10%, or about 1% to about 5% by weight of the secondcomposition. In certain embodiments, a viscosity increasing agent may bepresent in the second composition in an amount of about 0.1%, 0.25%,0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, or 30% by weight of the second composition or any range and/orindividual value therein.

Exemplary organic solvents for the second composition include, but arenot limited to, acetone, methyl alcohol, ethanol, isopropanol, butylalcohol, ethyl acetate, dimethyl isosorbide, propylene glycol, glycerol,ethylene glycol, polyethylene glycol, diethylene glycol monoethyl etheror mixtures thereof. In some embodiments of the present invention, theorganic solvent in the second composition may be ethanol and/orisopropyl alcohol. An organic solvent may be present in the secondcomposition in an amount of about 40% to about 90% by weight of thesecond composition or any range and/or individual value therein, suchas, but not limited to, about 40% to about 80%, about 50% to about 70%,about 50% to about 80%, about 60% to about 90%, about 70% to about 90%,or about 75% to about 85% by weight of the second composition. Incertain embodiments, an organic solvent may be present in the secondcomposition in an amount of about 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, or 90% by weight of the second composition or any range and/orindividual value therein.

Exemplary humectants for the second composition include, but are notlimited to, glycols, such as diethylene glycol monoethyl ether;glycerols; sugar polyols, such as sorbitol, xylitol and maltitol;polyols such as polydextroses; quillaia, urea, and blends thereof. Insome embodiments, the humectant in the second composition may comprisean alkylene glycol, such as, for example, hexylene glycol. A humectantmay be present in the second composition in an amount of about 2% toabout 20% by weight of the second composition or any range and/orindividual value therein, such as, but not limited to, about 2% to about15%, about 5% to about 15%, or about 15% to about 20% by weight of thesecond composition. In certain embodiments, a humectant may be presentin the second composition in an amount of about 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% byweight of the second composition or any range and/or individual valuetherein.

Exemplary water repellants for the second composition include, but arenot limited to, silicones, such as cyclomethicone, dimethicone,simethicone, C26-28 alkyl dimethicone, C26-28 alkyl methicone,polyphenylsisquioxane, trimethylsiloxysilicate and crosspolymers ofcyclopentasiloxane and dimethicone/vinyltrimethylsiloxysilicate, andblends thereof. In some embodiments, a second composition may comprisecyclomethicone. A water repellant may be present in the secondcomposition in an amount of about 0.5% to about 15% by weight of thesecond composition or any range and/or individual value therein, suchas, but not limited to, about 0.5 % to about 10%, about 1% to about 5%,or about 2% to about 5% by weight of the second composition. In certainembodiments, a water repellant may be present in the second compositionin an amount of about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, or 15% by weight of the second composition or anyrange and/or individual value therein.

Accordingly, a topical antiviral composition of the present inventionmay comprise at least one polyhydric alcohol, a first viscosityincreasing agent, water, a second viscosity increasing agent, at leastone organic solvent, at least one humectant, optionally at least onesolvent in addition to water, optionally an emollient, optionally awater repelling agent, optionally at least one preservative, andoptionally at least one buffering agent and/or buffer. The compositionmay be buffered to a pH of about 3 to about 11, such as, but not limitedto, about 6 to about 10, about 7 to about 10, or about 7 to about 9. Insome embodiments, the composition may have a pH of 7 or greater, 7.5 orgreater, or 9.5 or greater. In certain embodiments, the composition maycomprise at least one API, such as, but not limited to, a nitricoxide-releasing active pharmaceutical ingredient. In some embodiments,the NO-releasing API may be a diazeniumdiolate modified macromolecule.

A topical antiviral composition of the present invention may comprise afirst composition and a second composition as described herein. As thoseof skill in the art will recognize, the amount or concentration ofindividual components in a composition of the present invention may varydepending on the amount of the first composition and second compositionpresent in the composition (e.g., the ratio of the first composition andsecond composition present in the composition). In some embodiments, theratio of a first composition of the present invention to a secondcomposition in a composition of the present invention may be about 5:1or less, in further embodiments, about 4:1 or less, about 3:1 or less,about 2:1 or less, about 1:1 or less, about 0.5:1 or less, or about0.2:1 or less. In particular embodiments, the ratio may be about 3:1. Infurther embodiments, the ratio may be about 1:1.

In some embodiments, a composition of the present invention maycomprise, consist essentially of, or consist of a polyhydric alcohol inan amount of about 0.5% to about 10% by weight of the composition, afirst viscosity increasing agent in an amount of about 0.01% to about 3%by weight of the composition, water in an amount of about 30% to about50% by weight of the composition, at least one solvent in addition towater in an amount of about 0.5% to about 10% by weight of thecomposition, an emollient in an amount of about 0.5% to about 5% byweight of the composition, a buffer in an amount of about 0.01% to about10% by weight of the composition, a second viscosity increasing agent inan amount of about 0.01% to about 10% by weight of the composition, anorganic solvent in an amount of about 20% to about 45% by weight of thecomposition, a humectant in an amount of about 2% to about 10% by weightof the composition, a water repelling agent in an amount of about 0.1%to about 10% by weight of the composition, an NO-releasing API in anamount of about 0.5% to about 25% by weight of the composition,optionally a buffering agent in an amount of about 0.001% to about 1% byweight of the composition, optionally a preservative in an amount ofabout 0.001% to about 1% by weight of the composition, and optionally aneutralizing agent. The buffer, buffering agent and/or neutralizingagent may be present in an amount sufficient to provide the first partof the composition with a pH of about 3 to about 8. The composition mayhave a pH of less than about 11, such as, but not limited to, less thanabout 9.5, less than about 7, or less than about 6. In some embodiments,the composition may have a pH of about 4.5. The first and secondviscosity increasing agents may be the same and/or different. In certainembodiments, the first viscosity increasing agent may be acarboxypolymethylene and the second viscosity increasing agent may be acellulose, such as, but not limited to, hydroxypropyl cellulose. In someembodiments, the first viscosity increasing agent may be a carboxymethylcellulose or a salt thereof and the second viscosity increasing agentmay be a cellulose, such as, but not limited to, hydroxypropylcellulose. In some embodiments, the composition may be cosmeticallyelegant.

In some embodiments, a composition of the present invention maycomprise, consist essentially of, or consist of a polyhydric alcohol inan amount of about 1% to about 7% by weight of the composition, a firstviscosity increasing agent in an amount of about 0.1% to about 3% byweight of the composition, water in an amount of about 25% to about 40%by weight of the composition, at least one solvent in addition to waterin an amount of about 0.5% to about 10% by weight of the composition, anemollient in an amount of about 0.5% to about 5% by weight of thecomposition, a buffer in an amount of about 0.01% to about 10% by weightof the composition, a second viscosity increasing agent in an amount ofabout 0.1% to about 2% by weight of the composition, an organic solventin an amount of about 20% to about 45% by weight of the composition, ahumectant in an amount of about 2% to about 7% by weight of thecomposition, a water repelling agent in an amount of about 1% to about5% by weight of the composition, an NO-releasing API in an amount ofabout 5% to about 20% by weight of the composition, optionally abuffering agent in an amount of about 0.01% to about 0.2% by weight ofthe composition, optionally a preservative in an amount of about 0.01%to about 0.3% by weight of the composition, and optionally aneutralizing agent. The buffer, buffering agent, and/or neutralizingagent may be present in an amount sufficient to provide the first partof the composition with a pH of about 4 or about 6. The composition mayhave a pH of less than about 11, such as, but not limited to, less thanabout 9.5, less than about 7, or less than about 6. In some embodiments,the composition may have a pH of about 4.5. The first and secondviscosity increasing agents may be the same and/or different. In certainembodiments, the first viscosity increasing agent may be acarboxypolymethylene and the second viscosity increasing agent may be acellulose, such as, but not limited to, hydroxypropyl cellulose. In someembodiments, the first viscosity increasing agent may be a carboxymethylcellulose or a salt thereof and the second viscosity increasing agentmay be a cellulose, such as, but not limited to, hydroxypropylcellulose. In some embodiments, the composition may be cosmeticallyelegant. In certain embodiments, the composition may comprise acomposition as set forth in Table 2 and/or Table 13.

In some embodiments, a composition of the present invention maycomprise, consist essentially of, or consist of a polyhydric alcohol inan amount of about 1% to about 15% by weight of the composition, a firstviscosity increasing agent in an amount of about 0.01% to about 2.5% byweight of the composition, water in an amount of about 25% to about 50%by weight of the composition, at least one additional solvent (e.g., analcohol) in an amount of about 0.5% to about 10% by weight of thecomposition, an emollient in an amount of about 0.5% to about 5% byweight of the composition, a buffer in an amount of about 0.01% to about10% by weight of the composition, a second viscosity increasing agent inan amount of about 0.01% to about 10% by weight of the composition, anorganic solvent in an amount of about 20% to about 50% by weight of thecomposition, a humectant in an amount of about 2% to about 10% by weightof the composition, a water repelling agent in an amount of about 0.1%to about 10% by weight of the composition, an NO-releasing API in anamount of about 0.5% to about 25% by weight of the composition, andoptionally a preservative in an amount of about 0.001% to about 0.5% byweight of the first composition.

A composition of the present invention may comprise at least twodifferent viscosity increasing agents. One viscosity increasing agentmay be present in the first part of a composition of the presentinvention and the other viscosity increasing agent may be present in thesecond part of the composition. In some embodiments, a composition ofthe present invention comprises a carboxypolymethylene and a cellulose,such as, but not limited to, hydroxypropyl cellulose.Carboxypolymethylene may be present in a first composition of thepresent invention and the cellulose may be present in a secondcomposition, which may be combined to form a composition of the presentinvention. In some embodiments, a composition of the present inventioncomprises carboxymethyl cellulose sodium and a second cellulose, suchas, but not limited to, hydroxypropyl cellulose. Carboxymethyl cellulosesodium may be present in a first composition of the present inventionand the second cellulose may be present in a second composition, whichmay be combined to form a composition of the present invention. Acomposition of the present invention comprising at least two differentviscosity increasing agents may provide a cosmetically elegantcomposition comprising an API, such as, but not limited to, aparticulate API and/or an insoluble API (e.g., an aqueous and/ormoisture insoluble API, such as, for example, benzoyl peroxide).

A composition of the present invention may comprise an API,carboxymethyl cellulose sodium, and hydroxypropyl cellulose, and may bea cosmetically elegant composition. The composition may not be grittyand/or may have a reduced grittiness compared to the API in the absenceof a composition of the present invention. The composition may not betacky (i.e., sticky) and/or may have a reduced tackiness (i.e.,stickiness) compared to the API in the absence of a composition of thepresent invention. The composition may have a reduced and/or increasedstiffness (i.e., hardness) and/or may have an increased homogeneitycompared to the API in the absence of a composition of the presentinvention. In some embodiments, a composition of the present inventionmay comprise an API and may be a cosmetically elegant, homogeneouscomposition.

According to embodiments of the present invention, a topical antiviralcomposition may be provided in a kit. In some embodiments, a kit of thepresent invention may comprise a first composition and a secondcomposition as described herein that may be combined to form the topicalantiviral composition. The kit may separately store the first and secondcomposition. The kit may be configured to mix the two compositions upondispensing and/or for application to the skin of a subject in a desiredratio (e.g., 1:1, 2:1, etc.).

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXAMPLES Example 1

Groups of rabbits received anti-viral treatments at various doses asdescribed below in Table 1. The formulations for the anti-viraltreatments in Groups A-G are provided in Tables 2 and 3. Each of theformulations for Groups A-E included two separate compositions that wereseparately stored in a dual chamber pump. Prior to application, the twocompositions were dispensed and mixed together in a 1:1 ratio to providea combined composition that was applied to the rabbit. The target pH forthe combined composition was pH 8.

TABLE 1 Anti-viral treatment dosages. Infection with CRPV (2 sites eachvirus) Group No. of Anti-viral treatments wt CRPV mE8-CRPV # rabbits(beginning on day 14) DNA DNA A 4 Placebo gel 5 ug 5 ug B 4 1%Nitricil ™ NVN1 5 ug 5 ug C 4 1.6% Nitricil ™ NVN4 5 ug 5 ug D 4 10.0%Nitricil ™ NVN1 5 ug 5 ug E 4 16.3% Nitricil ™ NVN4 5 ug 5 ug F 4Placebo ointment 5 ug 5 ug G 4 Single Phase, 10% 5 ug 5 ug Nitricil ™NVN1 Ointment H 4 Cidofovir (0.3% 5 ug 5 ug formulated in cremophor;positive control)

TABLE 2 Formulations for the anti-viral treatments in Groups A-E. % w/w1% 1.6% 10% 16.3% Placebo Nitricil ™ Nitricil ™ Nitricil ™ Nitricil ™Ingredient Gel NVN1 NVN4 NVN1 NVN4 Isopropyl Alcohol 42.75 41.75 41.1533.25 27.25 Hexylene Glycol 5.0 5.0 5.0 5.0 5.0 Cyclomethicone 1.25 1.251.25 1.25 1.25 Hydroxypropyl cellulose 1.0 1.0 1.0 0.5 0.5 Nitricil ™NVN1 Drug — 1.0 — 10.0 — Substance Nitricil ™ NVN4 Drug — — 1.6 — 16Substance Purified Water 39.65 40.65 39.65 40.65 39.65 Glycerin, USP 5.02.05 5.0 2.05 5.0 Potassium Phosphate 1.35 5.9 1.35 5.9 1.35 MonobasicPotassium Phosphate Dibasic 2.6 — 2.6 — 2.6 Carboxymethylcellulose 1.41.4 1.4 1.4 1.4 Sodium Total 100.0 100.0 100.0 100.0 100.0

TABLE 3 Formulations for the anti-viral treatments in Groups F and G. %w/w Placebo 10% Nitricil ™ Ingredient Ointment NVN1 Ointment Mineral Oil& Polyethylene 43.0 38.7 White Petrolatum 43.0 38.7 Medium ChainTriglycerides 8.0 7.2 Mineral Oil 4.0 3.6 Macrogol 6 Glycerol 2.0 1.8Caprylocaprate NVN1 Drug Substance — 10.0 Total 100.0 100.0

Rabbits were infected with wild-type cottontail rabbit papillomavirus(wt CRPV) and E8-knock-out CRPV (mE8-CRPV) beginning on day 1 for anearly therapeutic antiviral treatment study. FIG. 1 shows the outline ofexperimental infections. The mE8-CRPV was included as this genomegenerates smaller, slower-growing papillomas that are more clinicallysimilar to human papillomavirus infections.

A total of 32 Adult New Zealand White rabbits (including both genders)were purchased from Robinson, PA. and used in the experiment. Therabbits were quarantined and cleared (14 days). Each rabbit wasinoculated with wt CRPV (at 2 sites; 5 μg/site) and mE8-CRPV viral DNA(at 2 sites; 5 μg/site). CRPV viral DNA was used to generate papillomasand infection was developed via a delayed scarification technique(Cladel N. M., et al., J Virol Methods 2008; 148(1-2):34-39). Of the twosites inoculated with one of the viruses, one site received treatment(i.e., the left site (L1 or L2)) and the other site was untreated (i.e.,the right site (R1 or R2)).

The rabbits were placed into one of eight groups (Groups A-H). A placebogroup (i.e., Group A) served as a control to assess local effects oftreatment in treated Groups B-H. Groups B-G represented test compoundcomparisons vs. placebo negative control.

Treatments for Groups A-H began at week two at a time when thepapillomas were not yet visible. This time point allowed for effects onsubclinical papillomas to be assessed. Treatment was 5× weekly(Monday-Friday), for five weeks with a dose of 0.1 ml per approximately2.5 cm×2.5 cm site for topical treatments. Body weights were takenweekly, and blood sera were collected at the end of the treatment periodfor blood chemistries, as needed.

The frequency and size of papillomas was measured weekly in 3 axes(length×width×height) in mm. Data was entered into a spread sheet andcalculations were conducted of the geometric mean diameter of eachpapilloma, mean±SEM for each group, Student's t-test between each pairedgroups and plots made of papilloma size vs time. Plots of weight changeswere also conducted.

At termination, kidney and liver samples were retrieved for histologyand toxicity assessment, as needed. Skin/papilloma sites were monitoredphotographically and biopsies assessed for histology atexperiment/treatment termination.

FIGS. 2-9 show plots of mean (±SEM) of the GMD measurements for eachtreatment group plotted against time after CRPV infection. Forcalculations of mean GMDs, the spontaneous regressor in Group D was notused. FIG. 10 shows mean body weights for the treatment groups.

As seen in FIG. 2, the gel vehicle without a NO-releasing API providedlittle to no separation in papilloma size between the treated anduntreated sites for either the wild type or mE8-CRPV mutant papillomavirus sites. In FIG. 3, the 1% Nitricil™ NVN1 group showed someseparation between treated and untreated sites for the mutant strain.However, complete inhibition of papilloma growth was not achieved. InFIG. 4, the 1.6% Nitricil™ NVN4 group showed little or no separationbetween treated and untreated sites for wild type virus and mutantstrain. FIG. 5 shows that 10% Nitricil™ NVN1 was effective in treatingboth wild type virus and the mutant strain with complete inhibition ofpapilloma growth in the mutant strain and substantial separation betweentreated and untreated sites in the faster growing wild type. FIG. 6shows that 16.3% Nitricil™ NVN4 was effective at inhibiting growth ofthe mutant strain but was not effective at inhibiting growth of the wildtype virus. As seen in FIG. 7, the ointment vehicle without aNO-releasing API provided little to no separation in papilloma sizebetween the treated and untreated sites for either the wild type ormutant papilloma virus sites. As seen in FIG. 8, the 10% NVN1 ointmentprovided little to no separation in papilloma size between the treatedand untreated sites for either the wild type or mutant papilloma virussites, illustrating the release of nitric oxide from the compositionaffects the effectiveness of inhibiting papilloma growth, as opposed tothe presence of the siloxane backbone as the 10% NVN1 ointment and the10% NVN1 gel both contained the same amount of the polysiloxane backbonebut achieved markedly different results.

Example 2

In vitro release testing was performed using multi-channel Nitric OxideAnalyzer. An analytical balance was used to weigh the test formulationsfrom Groups B-E and G described in Example 1. Approximately 20-mg ofboth phases of a respective formulation were transferred to a clean, dryNO measurement cell with a magnetic stir bar. The real time in vitrorelease of nitric oxide from the formulations was determined while undercontinuous mixing using the following instrumental parameters:

1. Moist Nitrogen Flow Rate: 112-115 ml/min

2. Sample Temperature: 37° C.

3. Detection: Nitric Oxide by Chemiluminescence

4. Data Acquisition Frequency: 1 Hz, Irregular Sequential Alternating

5. Duration: Time at which NO release rate decreases linearly (NLT 8 hr)

6. Acquisition Software: NovanWare

Conversion from parts per billion (PPB) NO to moles nitric oxide wasachieved by measuring the nitric oxide generated from a known amount ofsodium nitrite in a solution of potassium iodide to acquire aPPB-to-mole conversion factor. Any gaps in real time nitric oxiderelease data resulting from multichannel operation were filled in byusing a linear interpolation program. For any sample that was notmeasured to exhaustion of nitric oxide, a linear extrapolation to zerorelease of the last 5000 sec of release was performed. Real time nitricoxide release data was then integrated, resulting in a total nitricoxide accumulation curve. Nitric oxide release parameters such asC_(max) (i.e., the maximum concentration of NO released), T_(max) (i.e.,the time at which C_(max) is achieved), Cumulative Nitric Oxide Released(i.e., the sum of all data points per unit time), and Time to Half ofTotal Released (T₅₀) (i.e., the time at which 50% of the cumulative NOis released) can be calculated from both the real time and totalaccumulation nitric oxide release curves. All of the above calculationswere performed automatically in custom-built data processing software(NovanWare).

The results from in vitro release testing, along with the respective pHof the admixtures are summarized in Table 4 below.

TABLE 4 NO release data for the formulations tested 24 Hour CumulativeT₅₀ for 24 Real Time Release hours C_(max), T_(max) Release GroupFormulation (nmol/mg) (minutes) (pmol/mg) (pmol/mg) B 1% Nitricil ™ NVN188.8 11.3 156.1 at 3.6 6.7 at 0.5 hours minutes 2.3 at 1 hour 1.3 at 2hours 0.7 at 3 hours 0.2 at 4 hours C 1.6% Nitricil ™ NVN4 54.9 198 4.5at 12 3.1 at 0.5 hours minutes 2.6 at 1 hour 2.3 at 2 hours 1.5 at 3hours 1.1 at 4 hours D 10% Nitricil ™ NVN1 934.7 9.15 3107.5 at 1.2 57.7at 0.5 hours minutes 37.4 at 1 hour 14.2 at 2 hours 9.2 at 3 hours 5.1at 4 hours E 16.3% Nitricil ™ NVN4 310.8 420 13.0 at 2.4 8.9 at 0.5hours minutes 7.9 at 1 hour 6.3 at 2 hours 6.2 at 3 hours 5.6 at 4 hoursG 10% Nitricil ™ NVN1 178.6 582 5.8 at 690 0.9 at 0.5 hours Ointmentminutes 1.0 at 1 hour 0.9 at 2 hours 0.9 at 3 hours 4.5 at 4 hours

Example 3

The test articles in Example 2 were applied to the 2.5 cm×2.5 cm sitesof the rabbits of Example 1. Application of 0.1 mL of the test articlesto 6.25 cm² results in an in vitro assay release of NO/cm² as reflectedin Table 5.

TABLE 5 NO release per unit area data for the formulations testedCumulative Real Time Release C_(max), T_(max) Release Group Formulation(nmol/cm²) (pmol/cm²) (pmol/cm²) B 1% Nitricil ™ NVN1 878.5 at 0.5 hours2322.7 at 3.6 99.1 at 0.5 hours 1044.6 at 1 hour minutes 34.8 at 1 hour1226.9 at 2 hours 18.7 at 2 hours 1274.4 at 3 hours 9.8 at 3 hours1294.8 at 4 hours 2.5 at 4 hours 1230.8 at 24 hours C 1.6% Nitricil ™NVN4 85.4 at 0.5 hours 67.1 at 12 45.4 at 0.5 hours 164.4 at 1 hourminutes 39.1 at 1 hour 291.3at 2 hours 34.5 at 2 hours 386.5 at 3 hours21.6 at 3 hours 453.8 at 4 hours 15.6 at 4 hours 816.3 at 24 hours D 10%Nitricil ™ NVN1 8701.2 at 0.5 hours 3107.5 at 1.2 859.0 at 0.5 hours9975.1 at 1 hour minutes 556.5 at 1 hour 11141.0 at 2 hours 211.9 at 2hours 11730.8 at 3 hours 136.9 at 3 hours 12173.8 at 4 hours 76.5 at 4hours 13908.2 at 24 hours E 16.3% Nitricil ™ NVN4 241.9 at 0.5 hours193.8 at 2.4 132.6 at 0.5 hours 468.5 at 1 hour minutes 117.6 at 1 hour824.6 at 2 hours 93.7 at 2 hours 1163.5 at 3 hours 91.8 at 3 hours1484.1 at 4 hours 83.5 at 4 hours 4624.5 at 24 hours G 10% Nitricil ™NVN1 19.1 at 0.5 hours 82.8 at 690 13.2 at 0.5 hours Ointment 42.7 at 1hour minutes 13.6 at 1 hour 88.1 at 2 hours 12.3 at 2 hours 132.9 at 3hours 12.7 at 3 hours 457.6 at 4 hours 64.7 at 4 hours 2572.0 at 24hours

Real time and cumulative NO release profiles are illustrated in FIGS. 11through 17 as described above. Based on the effectiveness of theformulations for treatment groups D and E against the mutant strains,parameters for real time NO release that may be anti-viral may beidentified as illustrated in FIGS. 16A, 16B, and 17. Accordingly, insome embodiments of the present invention, the NO release from ananti-viral composition may fall within one or more of the windowsillustrated in FIGS. 16A and/or 17. The NO release within the definedwindows may occur at any time point within the anticipated time in whichthe composition will be applied. Accordingly, the NO release fallingwithin the window may occur within the first 1, 2, or 4 hours or mayoccur beginning at another time during the application period. The timewindows illustrated in FIGS. 16A, 16B, and 17 are shown in Table 6.

TABLE 6 NO release time windows. Min Real Max Real Window Duration TimeNO Time NO 0.5 Hour Weighted 0.5 hours 25 pmol/mg 4000 pmol/mg 1 HourWeighted 1 hour 7 pmol/mg 4000 pmol/mg 2 Hour Weighted 2 hours 6 pmol/mg4000 pmol/mg 3 Hour Weighted 4 hours 5 pmol/mg 4000 pmol/mg 1 Hour Area1 hour 74.4 nmol/cm² 59.52 nmol/cm² 2 Hour Area 2 hours 89.28 nmol/cm²59.52 nmol/cm² 3 Hour Area 4 hours 104.16 nmol/cm² 59.52 nmol/cm²

Example 4

Groups of rabbits received anti-viral treatments at various doses asdescribed below in Table 7. The formulations for the anti-viraltreatments in Groups A-F are provided in Table 8. Each of theformulations for Groups A-E included two separate compositions that wereseparately stored in a dual chamber pump. Prior to application, the twocompositions were dispensed and mixed together in a 1:1 ratio to providea combined composition that was applied to the rabbit. The target pH forthe combined composition was pH 8.

TABLE 7 Anti-viral treatment dosages. Infection with CRPV (2 sites eachvirus) Group No. of Anti-viral treatments wt CRPV mE8-CRPV # rabbits(beginning on day 14) DNA DNA A 4 Placebo 5 ug 5 ug B 4 2% Nitricil ™NVN1 5 ug 5 ug C 4 4% Nitricil ™ NVN1 5 ug 5 ug D 4 8% Nitricil ™ NVN1 5ug 5 ug E 4 10% Nitricil ™ NVN1 5 ug 5 ug F 4 Imiquimod control 5 ug 5ug

TABLE 8 Formulations for the anti-viral treatments in Groups A-E. % w/w2% 4% 8% 10% Nitricil ™ Nitricil ™ Nitricil ™ Nitricil ™ IngredientPlacebo NVN1 NVN1 NVN1 NVN1 Isopropyl Alcohol 42.75 40.75 38.75 34.7533.25 Hexylene Glycol 5.0 5.0 5.0 5.0 5.0 Cyclomethicone 1.25 1.25 1.251.25 1.25 Hydroxypropyl cellulose 1.0 1.0 1.0 1.0 0.5 Nitricil ™ NVN1Drug — 2.0 4.0 8.0 10.0 Substance Purified Water 39.65 40.65 40.65 40.6540.65 Glycerin, USP 5.0 2.05 2.05 2.05 2.05 Potassium Phosphate 1.35 5.95.9 5.9 5.9 Monobasic Potassium Phosphate Dibasic 2.6 — — — —Carboxymethylcellulose 1.4 1.4 1.4 1.4 1.4 Sodium Total 100.0 100.0100.0 100.0 100.0

TABLE 9 NO release data for the formulations of Table 8 Average T₅₀ ofRelease Cumulative Cumulative Rate Real Time Release Release C_(max),T_(max) nmol NO/ Release Formulation (nmol/mg) (minutes) (pmol/mg) mghour^(0.5) (pmol/mg) 2% Nitricil ™ 141.6 (at 3.81 398 at 1.8 628 over90.1 at 0.02 hours NVN1 2.5 hours) minutes 0.0784 56.25 at 0.1 hourhours 8.06 at 0.5 hours 3.37 at 1 hours 1.67 at 2 hours 4% Nitricil ™292.7 (at 4.15 1148 at 1.2 912 over 1147.8 at 0.02 hours NVN1 2.5 hours)minutes 0.0784 239.74 at 0.1 hour hours 18.0 at 0.5 hours 3.86 at 1hours 0.56 at 2 hours 8% Nitricil ™ 681.4 (at 4.25 2180 at 1.8 2831 over427.7 at 0.02 hours NVN1 4 hours) minutes 0.0784 524.4 at 0.1 hour hours29.73 at 0.5 hours 8.68 at 1 hours 1.79 at 2 hours 10% Nitricil ™ 905.2(at 3.50 2403 at 1.2 4516 over 2402 at 0.02 hours NVN1 4 hours) minutes0.0784 736.9 at 0.1 hour hours 414.6 at 0.5 hours 33.6 at 1 hours 13.8at 2 hours

Rabbits were infected with wild-type cottontail rabbit papillomavirus(wt CRPV) and E8-knock-out CRPV (mE8-CRPV) beginning on day 1 for anearly therapeutic antiviral treatment study. FIG. 1 shows the outline ofexperimental infections. The mE8-CRPV was included as this genomegenerates smaller, slower-growing papillomas that are more clinicallysimilar to human papillomavirus infections.

A total of 24 Adult New Zealand White rabbits (including both genders)were purchased from Robinson, PA. and used in the experiment. Therabbits were quarantined and cleared (14 days). Each rabbit wasinoculated with wt CRPV (at 2 sites; 5 μg /site) and mE8-CRPV viral DNA(at 2 sites; 5 μg /site). CRPV viral DNA was used to generate papillomasand infection was developed via a delayed scarification technique(Cladel N. M., et al., J Virol Methods 2008; 148(1-2):34-39). Of the twosites inoculated with one of the viruses, one site received treatment(i.e., the left site (L1 or L2)) and the other site was untreated (i.e.,the right site (R1 or R2)).

The rabbits were placed into one of six groups (Groups A-F). A placebogroup (i.e., Group A) served as a control to assess local effects oftreatment in treated Groups B-E.

Treatments for Groups A-F began at week two at a time when thepapillomas were not yet visible. This time point allowed for effects onsubclinical papillomas to be assessed. Treatment was 5× weekly(Monday-Friday), for five weeks with a dose of 0.1 ml per approximately2.5 cm×2.5 cm site for topical treatments. Body weights were takenweekly, and blood sera were collected at the end of the treatment periodfor blood chemistries, as needed.

The frequency and size of papillomas was measured weekly in 3 axes(length×width×height) in mm. Data was entered into a spread sheet andcalculations were conducted of the geometric mean diameter of eachpapilloma, mean +SEM for each group, Student's t-test between eachpaired groups and plots made of papilloma size vs time. Plots of weightchanges were also conducted.

At termination, kidney and liver samples were retrieved for histologyand toxicity assessment, as needed. Skin/papilloma sites were monitoredphotographically and biopsies assessed for histology atexperiment/treatment termination.

FIGS. 18-23 show graphs of papilloma size (GMD) in mm over time for theformulations in Groups A-F, respectively. In light of the dosing regimenof 5 days per week and once daily, doses that show only minimal efficacycould be suitable for use with a different dosing schedule, e.g., twicedaily 7 days per week. Based on the effectiveness of the formulationsfor treatment Groups D and E against the mutant strains, parameters forreal time NO release may fall within one or more of the windowsillustrated in FIGS. 16A and/or 17. However, other doses may also beeffective based on treatment Groups B and C as the duration of NOrelease for these groups may allow for more frequent dosing.Accordingly, a product (e.g., a topical composition) with an NO releasefalling within a 30 minute window defined as measured based on weightand having a minimum instantaneous release of 7 pmol/mg and a maximuminstantaneous release of 4,000 pmol/mg may be suitable. In someembodiments, a product with an NO release falling within a 30 minutewindow defined as measured based on weight and having a minimuminstantaneous release of 15 pmol/mg and a maximum instantaneous releaseof 4,000 pmol/mg may be suitable. The NO release within the definedwindows may occur at any time point within the anticipated time in whichthe product will be applied and/or present on the skin of a subject.Accordingly, an NO release falling within one or more of the windowsillustrated in FIGS. 16A and/or 17 may occur within the first 1, 2, or 4hours after application/administration or may occur beginning at anothertime after application/administration.

Example 5

Tissue samples from New Zealand white rabbits infected with CottontailRabbit Papillomavirus and treated as described in Example 4 wereobtained at the end of treatment. The skin sections were processed tohematoxylin and eosin (H & E) slides and submitted for microscopicevaluation in a blinded fashion. After evaluating the slides, theresults were summarized into three main categories: 1) papilloma, 2)hyperplasia of marked intensity, and 3) hyperplasia of minimal to mildintensity. The presence of inflammatory cells was determinedqualitatively. No quantitative analysis was performed. However, thequalitative assessment revealed a similar level of inflammation amongstthe three categories, which was that inflammation was generally low andcomparable. Table 10 provides the histology results from the H&Estaining.

TABLE 10 H&E Histology Results. Category Histopathological Findings #1-Papilloma Extensive exophytic proliferation of squamous epithelium whichis well differentiated. Finger-like papillary projections that protrudeover the surface of the epidermis covered by layers of keratin mixedwith serocellular material. High levels of intra-nuclear dark to lightbasophilic viral inclusions Diffuse infiltration of minimal to mildnumbers of inflammatory cells that include in decreasing order:lymphocytes, plasma cells, and heterophils with rare macrophages. #2-Hyperplasia Combination of exophytic and endophytic of marked intensityproliferation of squamous epithelium which is well differentiated.Papillary projections either protrude or invade within the dermis andare covered by moderate to marked degrees of keratin that are rarelymixed with serocellular material. Present within the squamous epitheliumare questionable intra-nuclear dark to light basophilic viralinclusions. However, the density of these inclusions is clearly fallless than the papillomas of Category #1 Mainly in the superficial dermisa more or less diffuse infiltration of mild numbers of inflammatorycells that include in decreasing order: lymphocytes, plasma cells, andheterophils with rare macrophages. #3- Hyperplasia Predominatelyendophytic proliferation of of minimal to mild squamous epithelium whichis well intensity differentiated. Papillary projections invade thedermis with a minimal amount of keratin covering the eipidermis.Intranuclear dark to light basophilic viral inclusion are not observed.Mainly in the superficial dermis a more or less diffuse infiltration ofmild numbers of inflammatory cells that include in decreasing order:lymphocytes, plasma cells, and heterophils with rare macrophages.

The tissues slides assigned to Category #3 (Hyperplasia of minimal tomild intensity) include the tissue samples obtained from animals treatedwith either 8% Nitricil™ NVN1 or 10% Nitricil™ NVN1. Intra-nuclear darkto light basophilic viral inclusions were not observed in the tissueslides assigned to Category #3, which suggests that treatment with highconcentrations of Nitricil™ NVN1, such as, for example, with the 8% or10% Nitricil™ NVN1 formulations, suppresses and/or inhibits viralreplication of a virus without altering the local infiltration ofinflammatory immune cells.

Example 6

Additional formulations were prepared and used to determine efficacy intreating and/or preventing virus-related cutaneous conditions, such as,for example, genital warts. These formulations included Nitricil™ NVN1in an amount of 4%, 8%, 12%, or 16% along with a placebo. Each of theformulations included a hydrogel having a pH of 4.5 and a composition asprovided in Table 11, and a second composition in the form of a gel andhaving a composition as provided in Table 12. Upon admixing the hydrogeland gel, a combined composition was achieved having a composition asprovided in Table 13.

TABLE 11 Composition of the pH 4.5 Hydrogel Component % w/w Water,Purified, USP 64.90 Potassium Phosphate Monobasic, NF 11.50 Alcohol, USP10.00 Glycerin, USP 8.00 Cyclomethicone, NF 3.00 CarboxymethylcelluloseSodium, NF 2.50 Benzoic Acid, USP 0.10 Total 100.00

TABLE 12 Composition of the Gel % w/w Component Placebo 8% 16% 24% 32%Isopropyl Alcohol, USP 85.45 78.50 70.50 62.75 54.75 Hexylene Glycol, NF10.00 10.00 10.00 10.00 10.00 Cyclomethicone, NF 2.50 2.50 2.50 2.502.50 Hydroxypropyl 2.00 1.00 1.00 0.75 0.75 cellulose, NF Nitricil ™NVN1 — 8.00 16.00 24.00 32.00 Titanium Dioxide, USP 0.05 — — — — Total100.00 100.00 100.00 100.00 100.00

TABLE 13 Composition of the combined composition % w/w Component Placebo4% 8% 12% 16% Isopropyl Alcohol, USP 42.725 39.25 35.25 31.375 27.375Water, Purified, USP 32.45 32.45 32.45 32.45 32.45 Potassium Phosphate5.75 5.75 5.75 5.75 5.75 Monobasic, NF Hexylene Glycol, NF 5.00 5.005.00 5.00 5.00 Alcohol (95% Ethanol), 5.00 5.00 5.00 5.00 5.00 USPNitricil ™ NVN1 — 4.00 8.00 12.00 16.00 Glycerin, USP 4.00 4.00 4.004.00 4.00 Cyclomethicone, NF 2.75 2.75 2.75 2.75 2.75Carboxymethylcellulose 1.25 1.25 1.25 1.25 1.25 Sodium, NF Hydroxypropyl1.00 0.50 0.50 0.375 0.375 Cellulose, NF Benzoic Acid, USP 0.05 0.050.05 0.05 0.05 Titanium Dioxide, USP 0.025 — — — — Total 100.00 100.00100.00 100.00 100.00

Example 7

Primary human keratinocytes were seeded on to a dermal equivalent andkept submerged in culture media for 2 days in order to establish cellmonolayers. After 2 days this entire assembly was lifted to theair:liquid surface, which corresponds to Day 0. On days 7-12, topicalapplication of 400 μL of Nitricil™ NVN1 or Nitricil™ NVN4 (in 50 mM PBS)was applied to the upper surface of the raft culture and incubated for 1hr. Following 1 hr incubation, the solution was gently aspirated fromthe surface of the raft cultures and the raft culture media wasreplenished.

Cultures were harvested on Day 13. Twelve hrs prior to harvest, cultureswere incubated with 100 μL/mL BrdU as a biomarker for cellular DNAsynthesis. Cultures were formalin-fixed and paraffin-embedded. Four μmsections were cut and stained with H&E. Adjacent sections were probedwith BrdU antibodies to determine the patterns and intensity of host DNAreplication.

Representative images of uninfected raft cultures treated with Nitricil™NVN1 and HPV-18 infected raft cultures treated with Nitricil™ NVN1 areprovided in FIGS. 24A-24C and 25A-25C, respectively. FIGS. 24A-24Cillustrate uninfected cultures that were treated with 0.5, 0.75, and 1.0mg/mL of Nitricil™ NVN1, respectively, and are stained with H&E orlabeled with BrdU antibodies. FIGS. 25A-25C illustrate HPV-18 infectedcultures that were treated with 0.75, 1.0, and 1.5 mg/mL of Nitricil™NVN1, respectively, and are stained with H&E or labeled with BrdUantibodies. The amount of HPV-18 viral replication in raft culturestreated with Nitricil™ NVN1 is provided in Table 14.

TABLE 14 HPV-18 viral copy number in raft cultures treated withNitricil ™ NVN1. Treatment % HPV-18 Copy Number* 50 mM PBS 100.00 0.75mg/mL Nitricil ™ NVN1 75.0 1.0 mg/mL Nitricil ™ NVN1 38.3 1.5 mg/mLNitricil ™ NVN1 14.95 Uninfected Raft Culture 0.02 *Percent HPV-18 CopyNumber compared to cultures treated with 50 mM PBS.

Representative images of uninfected raft cultures treated with Nitricil™NVN4 and HPV-18 infected raft cultures treated with Nitricil™ NVN4 areprovided in FIGS. 26A-26D and 27A-27D, respectively. FIGS. 26A-26Dillustrate uninfected cultures that were treated with 0.75, 1.0, 1.5,and 2.0 mg/mL of Nitricil™ NVN4, respectively, and are stained with H&Eor labeled with BrdU antibodies. FIGS. 27A-27D illustrate HPV-18infected cultures that were treated with 0.75, 1.0, 1.5, and 2.0 mg/mLof Nitricil™ NVN4, respectively, and are stained with H&E or labeledwith BrdU antibodies. The amount of HPV-18 viral replication in raftcultures treated with Nitricil™ NVN4 is provided in Table 15.

TABLE 15 HPV-18 viral copy number in raft cultures treated withNitricil ™ NVN4. Treatment % HPV-18 Copy Number* 50 mM PBS 100.00 0.75mg/mL Nitricil ™ NVN4 63.1 1.0 mg/mL Nitricil ™ NVN4 47.3 1.5 mg/mLNitricil ™ NVN4 29.3 2.0 mg/mL Nitricil ™ NVN4 14.8 Uninfected RaftCulture 0.02 *Percent HPV-18 Copy Number compared to cultures treatedwith 50 mM PBS.

The efficacy of topical nitric oxide-releasing candidates to inhibitviral replication was determined in vitro utilizing organotypic culturesof primary human keratinocytes containing HPV-18 genomic replicons. Theresults following 1 hr per day topical application of drug substance forsix days demonstrated a dose responsive reduction in viral DNA copynumber as determined by qPCR. This data supports a direct effect ofnitric oxide-releasing drug candidates on viral replication atconcentrations (1.5 or 2.0 mg/mL) that are not cytotoxic to host cellsand suggests that nitric oxide has a preferential deleterious effect onviral replication.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein. Allpublications, patent applications, patents, patent publications, andother references cited herein are incorporated by reference in theirentireties for the teachings relevant to the sentence and/or paragraphin which the reference is presented.

That which is claimed is:
 1. A method of treating and/or preventing aviral infection in a subject in need thereof comprising: administering atopical composition to the skin of a subject, wherein the topicalcomposition comprises a nitric oxide-releasing active pharmaceuticalingredient in an amount of about 0.5% to about 20% by weight of thecomposition, thereby treating and/or preventing the viral infection inthe subject.
 2. The method of claim 1, wherein the topical compositionreleases nitric oxide in an amount of about 0.05% to about 6% by weightof the composition, as measured by real time in vitro release testing.3. The method of any one of claim 1 or 2, wherein the topicalcomposition releases nitric oxide in a cumulative amount of about 10nmol of NO/mg of the composition to 1000 nmol of NO/mg of thecomposition in a time period of 1 hour after administration of thetopical composition to the skin of the subject, as measured by real timein vitro release testing.
 4. The method of any one of claims 1-3,wherein the topical composition releases nitric oxide in a cumulativeamount of about 90 nmol of NO/mg of the composition to 450 nmol of NO/mgof the composition in a time period of 4 hours after administration ofthe topical composition to the skin of the subject, as measured by realtime in vitro release testing.
 5. The method of any one of claims 1-4,wherein the topical composition releases nitric oxide in a cumulativeamount of about 180 nmol of NO/mg of the composition to 1000 nmol ofNO/mg of the composition in a time period of 24 hours afteradministration of the topical composition to the skin of the subject, asmeasured by real time in vitro release testing.
 6. The method of any oneof claims 1-5, wherein the topical composition provides a continuousrelease of nitric oxide for at least about 5 hours after administrationof the topical composition to the skin of the subject, as measured byreal time in vitro release testing.
 7. The method of claim 6, wherein,during the continuous release of nitric oxide, the topical compositionhas a release of NO that, on average, is in a range of about 1 to about500 pmol of NO/mg of the composition, as measured by real time in vitrorelease testing.
 8. The method of any one of claims 1-7, wherein thenitric oxide-releasing active pharmaceutical ingredient releases nitricoxide in an amount of at least about 50% in about 9 minutes or moreafter administration of the topical composition to the skin of thesubject, based on a total NO release determined at 24 hours afteradministration and measured by real time in vitro release testing. 9.The method of any one of claims 1-8, wherein the nitric oxide-releasingactive pharmaceutical ingredient releases nitric oxide in an amount ofat least about 50% in a time period of about 9 minutes to about 8 hoursafter administration of the topical composition to the skin of thesubject, based on a total NO release determined at 24 hours afteradministration and measured by real time in vitro release testing.
 10. Amethod of treating and/or preventing a viral infection in a subject inneed thereof comprising: administering a topical composition to the skinof a subject, wherein the topical composition comprises a nitricoxide-releasing active pharmaceutical ingredient that releases nitricoxide to the skin of the subject, and wherein the topical compositionmaintains a real time concentration of nitric oxide of at least about 7pmol of NO/mg of the composition for at least 1 hour afteradministration, as measured by real time in vitro release testing,thereby treating and/or preventing the viral infection in the subject.11. The method of claim 10, wherein the topical composition maintains areal time concentration of nitric oxide of at least about 6 pmol ofNO/mg of the composition for at least 2 hours after administration, asmeasured by real time in vitro release testing.
 12. The method of anyone of claim 10 or 11, wherein the topical composition maintains a realtime concentration of nitric oxide of at least about 5 pmol of NO/mg ofthe composition for at least 4 hours after administration, as measuredby real time in vitro release testing.
 13. The method of any one ofclaims 10-12, wherein the topical composition has a maximumconcentration of nitric oxide in a range of about 12 pmol of NO/mg ofthe composition to about 3500 pmol of NO/mg of the composition, asmeasured by real time in vitro release testing.
 14. The method of anyone of claims 10-13, wherein the topical composition releases nitricoxide in a cumulative amount of about 300 nmol of NO/mg of thecomposition to about 1000 nmol of NO/mg of the composition at 24 hoursafter administration, as measured by real time in vitro release testing.15. The method of any one of claims 10-14, wherein the topicalcomposition has a half life in a range of about 9 minutes to about 420minutes, as measured by real time in vitro release testing.
 16. A methodof treating and/or preventing a viral infection in a subject in needthereof comprising: administering a topical composition to the skin of asubject, wherein the topical composition comprises a nitricoxide-releasing active pharmaceutical ingredient that releases nitricoxide to the skin of the subject, and wherein the topical compositionmaintains a real time concentration of nitric oxide of at least about104 pmol of NO/cm² over a time period of at least 1 hour afteradministration of the composition to the skin of the subject, asmeasured by real time in vitro release testing, thereby treating and/orpreventing a viral infection in the subject.
 17. The method of claim 16,wherein the topical composition maintains a real time concentration ofnitric oxide of at least about 89 pmol of NO/cm² over a time period ofat least 2 hours after administration of the composition to the skin ofthe subject, as measured by real time in vitro release testing.
 18. Themethod of any one of claim 16 or 17, wherein the topical compositionmaintains a real time concentration of nitric oxide of at least about 74pmol of NO/cm² over a time period of at least 4 hours afteradministration of the composition to the skin of the subject, asmeasured by real time in vitro release testing.
 19. The method of anyone of claims 16-18, wherein the topical composition releases nitricoxide in a cumulative amount of about 1300 nmol of NO/cm² to about 14000nmol of NO/cm² in a time period of about 4 hours after administration ofthe composition to the skin of the subject, as measured by real time invitro release testing.
 20. The method of any one of claims 16-19,wherein the topical composition releases nitric oxide in a cumulativeamount of about 4500 nmol of NO/cm² to about 14000 nmol of NO/cm² in atime period of about 24 hours after administration of the composition tothe skin of the subject, as measured by real time in vitro releasetesting.
 21. The method of any one of claims 16-20, wherein, afteradministration of the topical composition to the skin of the subject,the topical composition has a real time concentration of NO of at least100 pmol of NO/cm² at 0.5 hours after administration, at least 50 pmolof NO/cm² at 1 hour after administration, at least 40 pmol of NO/cm² at2 hours after administration, at least 25 pmol of NO/cm² at 3 hoursafter administration, and/or at least 20 pmol of NO/cm² at 4 hours afteradministration, as measured by real time in vitro release testing. 22.The method of any one of claims 1-21, wherein the topical compositiondoes not comprise acidified nitrite.
 23. The method of any one of claims1-22, wherein the viral infection is caused by cytomegalovirus (CMV),epstein-barr virus, varicella zoster virus (VZV), vaccinia virus, cowpoxvirus, monkeypox virus, herpes simplex virus (HSV), herpes zoster, humanherpes virus 6 (HHV-6), human herpes virus 8 (HHV-8), papillomavirus,molluscum contagiosum, orf, variola, and/or coxsackie virus.
 24. Themethod of any one of claims 1-23, wherein the viral infection is causedby a papillomavirus
 25. The method of any one of claims 1-24, whereinthe viral infection is caused by herpes simplex type 1 and/or herpessimplex type
 2. 26. The method of any one of claims 1-25, wherein theviral infection infects the skin of the subject.
 27. The method of anyone of claims 1-26, wherein the method prevents a viral infection in thesubject.
 28. The method of any one of claims 1-27, wherein the methodtreats a viral infection in the subject.
 29. The method of any one ofclaims 1-28, wherein the method prevents and/or reduces the appearanceand/or size of a benign lesion (e.g., a wart).
 30. The method of any oneof claims 1-29, wherein the method prevents and/or reduces theappearance and/or size of a malignant lesion.
 31. The method of any oneof claims 1-30, wherein the subject is a human.
 32. The method of anyone of claims 1-31, wherein the administering step comprises applyingthe topical composition to virally infected skin of the subject.
 33. Themethod of claim 32, wherein the virally infected skin comprises a benignlesion.
 34. The method of claim 33, wherein the virally infected skincomprises a wart, sore, papilloma, blister, and/or rash.
 35. The methodof claim 33, wherein the virally infected skin comprises a wart and themethod further comprises debriding the wart prior to administering thetopical composition to the skin of the subject.
 36. The method of claim33, wherein the virally infected skin comprises a wart and the method donot comprise debriding the wart prior to administering the topicalcomposition to the skin of the subject.
 37. The method of any one ofclaims 1-36, wherein the nitric oxide-releasing active pharmaceuticalingredient comprises a nitric oxide-releasing compound having adiazeniumdiolate functional group.
 38. The method of claim 37, whereinthe nitric oxide releasing compound comprises a NO-releasingco-condensed silica particle.
 39. The method of any one of claims 1-38,wherein the topical composition has a pH in a range of about 5 to about10.
 40. The method of any one of claims 1-39, wherein the topicalcomposition has a pH in a range of about 6 to about
 8. 41. The method ofany one of claims 1-40, wherein the topical composition comprises: afirst viscosity increasing agent; at least one polyhydric alcohol; atleast one buffer; a second viscosity increasing agent; at least oneorganic solvent; at least one humectant; at least one activepharmaceutical ingredient; and water.
 42. The method of any one ofclaims 1-41, wherein the topical composition comprises two compositionsthat are mixed together prior to and/or during the administering step.43. The method of any one of claims 1-42, wherein a treatment effectiveand/or a prevention effective amount of nitric oxide is administered tothe basal layer and/or basement membrane of a subject's epithelium. 44.The method of any one of claims 1-43, wherein nitric oxide in an amountof about 1×10⁻⁵ M to about 1×10⁻⁷ M is administered to the basal layerand/or basement membrane of a subject's epithelium.
 45. The method ofany one of claims 1-44, wherein nitric oxide is administered in anamount sufficient to induce apoptosis in virally infected cells.
 46. Themethod of any one of claims 1-44, wherein viral replication is reducedand/or eliminated with less than about 50% host cell cytotoxicity. 47.Use of a topical composition comprising a nitric oxide-releasing activepharmaceutical ingredient in an amount of about 0.5% to about 20% byweight of the composition to treat and/or prevent a viral infection in asubject in need thereof
 48. Use of the topical composition of claim 47,wherein viral replication is reduced and/or eliminated with less thanabout 50% host cell cytotoxicity.